Purpose: The genus Pseudomonas is a ubiquitous microorganism frequently detected from immunocompromised patients. The inherent resistance to numerous antimicrobial agents contributes to the opportunistic character of ...Purpose: The genus Pseudomonas is a ubiquitous microorganism frequently detected from immunocompromised patients. The inherent resistance to numerous antimicrobial agents contributes to the opportunistic character of this pathogen exhaustive monitoring of this pathogen is considered of critical importance to public health organizations. The reliable identification method able to distinguish genetic close Pseudomonas species is needed, because these organisms are difficult to differentiate by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect four Pseudomonas species which are frequently detected from the human oral cavities, and to investigate the distribution of these organisms in the living environment using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the rpoD gene of four Pseudomonas species. Swab samples were collected from fifty washstands, and the distribution of Pseudomonas species was investigated using a conventional PCR at genus level and a multiplex PCR at species level. Results: Multiplex PCR method developed in this study was able to distinguish four Pseudomonas species clearly. The genus Pseudomonas was detected from all samples (100%), whereas P. putida, P, aeruginosa, P. stutzeri and P. fluorescens were detected at 44%, 8%, 4% and 2% in fifty swab samples, respectively. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction. It was indicated that washstands were the uninhabitable environment for P. putida, P, aeruginosa, P. stutzeri and P. fluorescens.展开更多
Purpose: In recent years, multidrug-resistant Acinetobacter baumannii has appeared and caused outbreaks of hospital infections all over the world. Close monitoring of this pathogen and other A. baumanii complex specie...Purpose: In recent years, multidrug-resistant Acinetobacter baumannii has appeared and caused outbreaks of hospital infections all over the world. Close monitoring of this pathogen and other A. baumanii complex species is considered of critical importance to public health organizations. The reliable identification method able to distinguish A. baumannii from genetically close Acinetobacter species is needed, because these species are unable to be differentiated by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect A. baumanii complex species, and Acinetobacter lwoffii which is frequently detected from the human specimens, and to investigate the distribution of these organisms in dental hospital using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S ribosomal RNA (16S rRNA) gene and DNA gyrase subunit B (gyrB) of each species of A. baumanii complex species. Swab samples were collected from ten dental spittoon units in dental hospital, and the distribution of A. baumanii complex species was investigated using a multiplex PCR. Results: These primers were able to distinguish each species of A. baumanii complex species clearly. A. baumanii and A. calcoaceticus were detected at 20.0% and 10.0% in ten swab samples, respectively. On the other hand, A. nosocomialis, A. lowffii, and A. pittii were detected from no sample. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.展开更多
Purpose: Recently, Candida haemulonii complex (Candida haemulonii, Candida duobushaemulonii and Candida haemulonii var. vulnera) and two genetically close species (Candida pseudohaemulonii and Candida auris) have emer...Purpose: Recently, Candida haemulonii complex (Candida haemulonii, Candida duobushaemulonii and Candida haemulonii var. vulnera) and two genetically close species (Candida pseudohaemulonii and Candida auris) have emerged as an opportunistic fungal pathogen associated with various infectious diseases of humans, and most of those isolates have displayed antifungal resistance. Because it is difficult to differentiate these microorganisms by a current technique, unfortunately, it is important to establish a method for identifying them accurately. The purpose of the present study was to design species-specific primers in order to identify and detect C. auris, C. pseudohaemulonii, and C. haemulonii complex, i.e. , C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 26S rRNA, 18S rRNA, and RPB1 genes and ITS region of five Candida species. Results: The multiplex PCR method developed in this study was able to distinguish five Candida species clearly. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction.展开更多
Cassava mosaic disease (CMD) caused by Cassava Mosaic Begomoviruses (CMBs) is one of the most devastating crop diseases and a major constraint for cassava production. In order to ensure surveillance for epidemic preve...Cassava mosaic disease (CMD) caused by Cassava Mosaic Begomoviruses (CMBs) is one of the most devastating crop diseases and a major constraint for cassava production. In order to ensure surveillance for epidemic prevention, low-cost diagnostic tools are appropriate for large-scale testing of cassava viruses. Multiplex PCR diagnosis is one approach that can reduce diagnostic costs and delays. A multiplex PCR approach was developed for simultaneous detection of African cassava mosaic virus (ACMV), East African Cassava Mosaic Virus and East African cassava mosaic Cameroon virus (EACMV/CM) in Togo CMD-infected cassava leaves. Three primers pairs were used to target their respective viruses in a single tube PCR. Multiplex PCR detected ACMV, EACMV and EACMV/CM in plant DNA extracts prepared from cassava leaves infected with CMB. The primers amplified 783 bp specific to ACMV, 650 bp specific to EACMV and 560 bp specific to EACMCV/CM in both uniplex and multiplex formats. Multiplex PCR is an excellent tool for the effective control of cassava diseases. .展开更多
PCRs based on different genes of Actinobacillus pleuropneumoniae have been developed for detecting and identifying A. pleuropneumoniae. Some of them could amplify positive fragments from the phylogenetically closely r...PCRs based on different genes of Actinobacillus pleuropneumoniae have been developed for detecting and identifying A. pleuropneumoniae. Some of them could amplify positive fragments from the phylogenetically closely related species bacteria. To improve veracity and specificity of PCR, a species-specific multiplex PCR assay was developed to identify and detect A. pleuropneumoniae, based on the 3′-terminus of the species-specific apxIVA gene and the already existing species-specific primers in the omlA gene. Both 346-bp and 950-bp fragments could be simultaneously amplified from all A. pleuropneumoniae reference strains and isolates, and the species specificity of the assay was evaluated with a collection of ten strains representing eight different species bacteria including species normally found in the respiratory tracts of swine. All of these strains turned out negative in the multiplex PCR. All sequences of products of multiplex PCR randomly sampled were also correct. The sensitivity of the multiplex PCR was determined to be 10 pg of A. pleuropneumoniae DNA. The multiplex PCR and bacterial isolation were compared to determine their sensitivities by using experimentally infected pigs and clinical disease pigs. The multiplex PCR was more sensitive than bacterial isolation. The multiplex PCR was also evaluated on mixed bacterial cultures from clinical healthy pigs. 26/100 (26%) of the subclinically infected pigs were detected from clinical healthy pigs. The results indicate that the multiplex PCR assay is a sensitive, highly specific, and effective diagnostic tool for identification and detection of A. pleuropneumoniae.展开更多
Objective:To evaluate microscopy,OptiMAL<sup>?</sup> and multiplex PCR for the identification of Plasmodium falciparum（P.falciparum） and Plasmodium vivax（P.vivax） from the field isolates of Bikaner,Raj...Objective:To evaluate microscopy,OptiMAL<sup>?</sup> and multiplex PCR for the identification of Plasmodium falciparum（P.falciparum） and Plasmodium vivax（P.vivax） from the field isolates of Bikaner,Rajasthan（Northwest India）.Methods:In this study,a multiplex PCR（P.falciparum and P.vivax） was further developed with the incorporation of Plasmodium malariae（P.malariae） specific primer and also a positive control.The performance of microscopy,plasmodium lactate dehydrogenase（pLDH） based malaria rapid diagnostic test OptiMAL<sup>?</sup> and 18S rRNA gene based multiplex PCR for the diagnosis of P.falciparum and P.vivax was compared.Results:The three species multiplex PCR if.falciparum,P.vivax and P.malariae) with an inbuilt positive control was developed and evaluated.In comparison with multiplex PCR,which showed the sensitivity and specificity of 99.36%（95%CI,98.11%-100.00%） and 100.00%（95%CI,100.00%-100.00%）,the sensitivity and specificity of microscopy was 90.44%（95%CI,88.849-95.04%） and 99.22%（95% CI,97.71%-100.00%）,and OptiMAL<sup>?</sup> was 93.58%（95%CI,89.75%-97.42%） and 97.69%（95%CI, 95.10%-100.00%）.The efficiencies were 99.65%,95.10%and 95.45%for multiplex PCK.microscopy and OptiMAL<sup>?</sup>.respectively.Conclusions:Our results raise concerns over the overall sensitivities of microscopy and OptiMAL<sup>?</sup>,when compared to the multiplex PCR and thus stress the need for new molecular interventions in the accurate detection of the malarial parasites.This further highlights the fact that further developments are needed to improve the performance of rapid diagnostic tests at field level.展开更多
Objective: To develop the multiplex PCR method based on the internal transcribed spacer 2 to discriminate the intestinal trematodes, Centrocestus caninus(C. caninus), and Stellantchasmus falcatus(S. falcatus).Methods:...Objective: To develop the multiplex PCR method based on the internal transcribed spacer 2 to discriminate the intestinal trematodes, Centrocestus caninus(C. caninus), and Stellantchasmus falcatus(S. falcatus).Methods: Four species of heterophyid trematodes including C. caninus, S. falcatus,Haplorchis taichui and Haplorchoides sp. were amplified and the specific primer was designed based on the internal transcribed spacer 2 region. Two specific primers were used to validate the optimized PCR conditions: the specificity test and the sensitivity test.Results: Both of these specific primers confirmed the specificity through multiplex PCR reaction which generated both PCR products(231 and 137 bp) in the mixed DNA template of C. caninus and S. falcatus with no cross-reaction with other heterophyid trematodes. The optimum annealing temperature of both primers was 54–59℃. The sensitivity test used the two-fold serial dilution DNA template, which was concentrated between 10 and 0.312 5 ng/mL. The lowest concentration of the DNA template of this multiplex PCR was 2.5 ng/mL.Conclusions: The technique described here proved to be a species-specific technique and was found to be a rapid method for the diagnosis of C. caninus and S. falcatus in terms of the larval and adult stages in intermediate and/or definitive hosts in the endemic area.展开更多
Recently, the incidence of<span> </span><i><span>Candida</span></i><span> infections has substantially increased. Conventional identification methods for </span><...Recently, the incidence of<span> </span><i><span>Candida</span></i><span> infections has substantially increased. Conventional identification methods for </span><i><span>Candida</span></i><span> species are technically difficult to conduct and cannot accurately distinguish each species. The purpose of the present study was to design primers to identify and detect simultaneously</span><span> </span><span>eight medically important </span><i><span>Candida</span></i><span> species using one-step multiplex PCR. PCR primers were designed based on partial sequences of intergenic spacer (IGS) and internal transcribed spacer (ITS) genes of eight medically important </span><i><span>Candida</span></i><span> species. These primers were able to distinguish each </span><i><span>Candida</span></i><span> species and did not display cross-reactivity with representative </span><i><span>Candida </span></i><span>species other than the eight</span><i><span> Candida</span></i><span> species. Moreover, our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.</span>展开更多
Hypertrophic cardiomyopathy (HCM) is one of the diseases damaging people health most badly and some mutations of exons in cardiac troponin I (cTnI) gene are closely associated with family hypertrophic cardiomyopathy (...Hypertrophic cardiomyopathy (HCM) is one of the diseases damaging people health most badly and some mutations of exons in cardiac troponin I (cTnI) gene are closely associated with family hypertrophic cardiomyopathy (FHCM).A microarray was fabricated to screen mutations in exons 3,5,7,and 8 in cTnI gene.Primers were designed for the PCR (polymerase chain reaction) to amplify the target DNA fragments from fresh blood samples.In order to simplify the PCR process,multiplex PCR technology was investigated in detail.The concentration of Mg^(2+) played an important role in multiplex PCR process,a properly low concentration of Mg^(2+) submitted a better speciality of PCR products.The speciality was also favored when the annealing temperature was reasonably enhanced and 64℃is the optimal annealing temperature for the multiplex PCR systems.When applying the fabricated gene-chip to detect the target fragments from PCR mixture,the signal intensity sequence is in accordance with that from theoretic estimate.展开更多
Objective:To develop a new technique for diagnosis of Plasmodium knowlesi and at the same time to be able to discriminate among the diverse species of Plasmodium causing human malaria.Methods:In this study the nested ...Objective:To develop a new technique for diagnosis of Plasmodium knowlesi and at the same time to be able to discriminate among the diverse species of Plasmodium causing human malaria.Methods:In this study the nested multiplex malaria PCR was redesigned,targeting the 18S rR NA gene,to identify the fifth human Plasmodium species,Plasmodium knowlesi,together with the other human Plasmodium(Plasmodium falciparum,Plasmodium vivax,Plasmodium ovale and Plasmodium malariae)by amplified fragment size using only two amplification processes and including an internal reaction control to avoid false negatives.Results:The technique was validated with 91 clinical samples obtained from patients with malaria compatible symptoms.The technique showed high sensitivity(100%)and specificity(96%)when it was compared to the reference method employed for malaria diagnosis in the Instituto de Salud Carlos栿and a published real-time PCR malaria assay.Conclusions:The technique designed is an economical,sensitive and specific alternative to current diagnosis methods.Furthermore,the method might be tested in knowlesi-malaria endemic areas with a higher number of samples to confirm the quality of the method.展开更多
Iwagaki oyster,Crassostrea nippona,widely distributes along the seashore of Eastern Asia,and has been considered to be a potential breeding species due to its delicious taste and high commercial value.In order to stud...Iwagaki oyster,Crassostrea nippona,widely distributes along the seashore of Eastern Asia,and has been considered to be a potential breeding species due to its delicious taste and high commercial value.In order to study its genetic background and population structure,we developed 46 novel polymorphic microsatellite markers using next-generation sequencing technique and characterized them in 30 individuals.The number of alleles ranged from 3 to 22,while the observed and expected heterozygosities varied from 0.133 to 1.000 and 0.455 to 0.949,respectively.Fifteen microsatellite markers were selected and grouped into five highly informative multiplex PCRs for C.nippona.We evaluated and validated these multiplex PCRs in a cultured population including 173 candidate parents and 486 offspring.In actual parentage analysis,80%of the offspring were correctly assigned to their parental pairs using three multiplex PCRs.Furthermore,the success rate of parentage assignment reached 96%when the other two multiplex PCRs were added.These 46 microsatellite loci with high variability and the five multiplex PCRs described here provide a powerful tool for pedigree reconstruction,resource conservation and selective breeding program of C.nippona.展开更多
Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system,different primer ratios and annealing temperatures in SSR marker amplification.Co...Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system,different primer ratios and annealing temperatures in SSR marker amplification.Concentration and gradient experiments for four components(enzyme,MgCl2,DNA template and dNTPs) in PCR system were used in the research with the concentration of the other component remained the same;the orthogonal design L9(34) was applied in the optimization of four sets of primers(STM0014,Pat,SSI,and UGP) in the reaction system at three levels;the temperature gradient selection was used to find out the optimum annealing temperature for the primer.The optimized multiplex PCR system of potato SSR marker with a total volume of 20 μL:2.5 μL 25 mmol · L-1 MgCl2,0.6 μL 10 mmol · L-1 dNTPs,0.8 U Taq,80 ng DNA template was ultimately established through the comparison and analysis of test results;the ratio of four pairs of 4 mmol · L-1 primers was 2:1:2:3,and the annealing temperature was 54.7℃.The optimized reaction system could be repeated stably;and the stable and reliable amplification results were able to clearly distinguish different potato varieties.This research built the solid foundation for the further study of genetic diversity of potato germplasms and construction of DNA fingerprinting..展开更多
Purpose: Although the genus Candida is frequently isolated from human oral cavities, the distribution at the species level of these organisms has been little reported. The purpose of the present study was to assess th...Purpose: Although the genus Candida is frequently isolated from human oral cavities, the distribution at the species level of these organisms has been little reported. The purpose of the present study was to assess the distribution at the species level of the genus Candida in human oral cavities. Methods: This study was performed using culture and Multiplex PCR methods. Moreover, the genotyping classification of C. albicans was analyzed with a PCR. Results: Of all subjects (n = 90), detection frequency of genus Candida was 42.2%. Genus Candida was not detected in the subjects between 0 to 9 years old, and there was no difference in the detection frequencies of this organism among each generation from 10s to 80s. C. albicans was the most dominant species, followed by C. parapsilosis, C. glabrata, and C. dubliniensis. Plural Candida species tended not to be detected in the individual sample. Genotype A was dominant in the C. albicans isolates. Conclusion: These results indicated that C. albicans of genotype A was dominant and that the genus Candida rarely coexists with other Candida species, in each individual oral cavity.展开更多
Purpose: Fusobacterium nucleatum is an opportunistic pathogen involved in periodontal diseases, extraoral infections, and colorectal cancer. Fusobacterium necrophorum causes a variety of necrotic infections. F. nuclea...Purpose: Fusobacterium nucleatum is an opportunistic pathogen involved in periodontal diseases, extraoral infections, and colorectal cancer. Fusobacterium necrophorum causes a variety of necrotic infections. F. nucleatum and F. necrophorum are classified into five and two subspecies, respectively. Conventional identification methods were technically hard to distinguish each subspecies of two Fusobacterium species accurately. The purpose of the present study was to design primers to identify two medically important Fusobacterium species at the subspecies level, using one-step multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S ribosomal RNA (16S rRNA) gene, RNA polymerase B (rpoB) gene, and DNA gyrase subunit B (gyrB) of each subspecies of F. nucleatum and F. necrophorum. Results: These primers were able to distinguish each subspecies of F. nucleatum and F. necrophorum and did not display cross-reactivity with representative Fusobacterium species other than F. nucleatum and F. necrophorum. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.展开更多
Objective To establish multiplex PCR-based assays for detecting H.influenzae and H.parainfluenzae.And the PCR-based assays were applied to detect the carriage rates of H.influenzae and H.parainfluenzae in nasopharynge...Objective To establish multiplex PCR-based assays for detecting H.influenzae and H.parainfluenzae.And the PCR-based assays were applied to detect the carriage rates of H.influenzae and H.parainfluenzae in nasopharyngeal swab specimens which were collected from healthy children.Methods Multiplex primers for species-specific PCR were designed by using DNAstar soft based on the sequences of 16S rRNA genes from genus Haemophilus to detect H.influenzae and H.parainfluenzae.Results The sensitivity of the 16S rRNA PCR assay for detecting H.influenzae and H.parainfluenzae was 97.53% and 100% respectively,and the specificity was 95.89% and 96.63% respectively.Youden's Index on the ability to detect H.influenzae and H.parainfluenzae was 0.9342 and 0.9663 respectively.666 nasopharyngeal swab specimens were collected from healthy children.The detection rates of H.influenzae and H.parainfluenzae were 14.11% and 16.07% respectively by using isolation and culture methods.The detection rates of H.influenzae and H.parainfluenzae were 43.54% and 57.96% respectively by 16S rRNA PCR assays.The carriage rates of serotypes a,b,c,d,e,f and non-typeable isolates were 0% (0/666),0.15% (1/666),1.20% (8/666),0.15% (1/666),1.20% (8/666),1.80% (12/666),95.50% (636/666) respectively.Conclusion The multiplex PCR assays were very rapid,reliable and feasible methods for detection of H.influenzae and H.parainfluenzae in pharyngeal swab specimens which were compared to conventional isolation and culture methods.95.5% of H.influenzae strains in healthy children were nontypeable.The encapsulated or typable strains were mainly three serotypes which was c,e,and f serotype.展开更多
Objective Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respir...Objective Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction(PCR) and capillary electrophoresis(MPCE) assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebacterium diphtheriae, and Streptococcus pyogenes. Methods Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. Results The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10^(-7) to 10^(-2) ng/μL. Furthermore, analysis of the 152 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. Conclusion This study revealed that the MPCE assay is a rapid, reliable, and high-throughput method with high specificity and sensitivity. This assay has great potential in the molecular epidemiological survey of respiratory pathogens.展开更多
There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical ...There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.展开更多
Objective To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray. Methods Two sets of the multiplex PCR system were...Objective To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray. Methods Two sets of the multiplex PCR system were used to amplify the target genes in genetically modified (GM) soybean. Seventeen capture probes (PCR products) and 17 pairs of corresponding primers were designed according to the genetic characteristics of Rroundup Ready soybean (GTS40-3-2), maize (Mon810, Nk603, GA21), canola (T45, MS1/RF1), and rice (SCK) in many identified GM crops. All of the probes were categorized and identified as species-specific probes. One negative probe and one positive control probe were used to assess the efficiency of all reactions, and therefore eliminate any false positive and negative results. After multiplex PCR reaction, amplicons were adulterated with Cy5-dUTP and hybridized with DNA microarray. The array was then scanned to display the specific hybridization signals of target genes. The assay was applied to the analysis of sample of certified transgenic soybean (Roundup Ready GTS40-3-2) and canola (MS1/RF1). Results A combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS1/RF1 canola with a great specificity and reliability. Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events, indicating a high sensitivity. Conclusion A combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.展开更多
Staphylococcus aureus, Escherichia coli and Bacillus cereus are the major agents of cow endometritis in dairy cows. A multiplex PCR (SEB-mPCR) was established based on the conserved genes of S. aureus, E. coli and B. ...Staphylococcus aureus, Escherichia coli and Bacillus cereus are the major agents of cow endometritis in dairy cows. A multiplex PCR (SEB-mPCR) was established based on the conserved genes of S. aureus, E. coli and B. cereus, and the detection limits were 103, 102 and 103 CFU mL-1, respectively. SEB-mPCR could not amplify genomic DNA of pathogenic bacteria of other common bovine diseases. A total of 309 vaginal discharge samples from cows with endometritis were tested by SEB-mPCR. Of the samples, 23.95% had the three kinds of bacteria detected, 17.15% had S. aureu and E. coli, 9.39% had E. coli and B. cereus, and 9.71% had S. aureus and B. cereus. The rates of infections with S. aureus, E. coli and B. cereus were 11.35, 16.18 and 9.06%, respectively. Therefore, SEB-mPCR has a potential as a diagnosis tool for endometritis in dairy cows.展开更多
文摘Purpose: The genus Pseudomonas is a ubiquitous microorganism frequently detected from immunocompromised patients. The inherent resistance to numerous antimicrobial agents contributes to the opportunistic character of this pathogen exhaustive monitoring of this pathogen is considered of critical importance to public health organizations. The reliable identification method able to distinguish genetic close Pseudomonas species is needed, because these organisms are difficult to differentiate by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect four Pseudomonas species which are frequently detected from the human oral cavities, and to investigate the distribution of these organisms in the living environment using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the rpoD gene of four Pseudomonas species. Swab samples were collected from fifty washstands, and the distribution of Pseudomonas species was investigated using a conventional PCR at genus level and a multiplex PCR at species level. Results: Multiplex PCR method developed in this study was able to distinguish four Pseudomonas species clearly. The genus Pseudomonas was detected from all samples (100%), whereas P. putida, P, aeruginosa, P. stutzeri and P. fluorescens were detected at 44%, 8%, 4% and 2% in fifty swab samples, respectively. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction. It was indicated that washstands were the uninhabitable environment for P. putida, P, aeruginosa, P. stutzeri and P. fluorescens.
文摘Purpose: In recent years, multidrug-resistant Acinetobacter baumannii has appeared and caused outbreaks of hospital infections all over the world. Close monitoring of this pathogen and other A. baumanii complex species is considered of critical importance to public health organizations. The reliable identification method able to distinguish A. baumannii from genetically close Acinetobacter species is needed, because these species are unable to be differentiated by phenotypic or biochemical methods. The purpose of the present study was to design species-specific primers in order to identify and detect A. baumanii complex species, and Acinetobacter lwoffii which is frequently detected from the human specimens, and to investigate the distribution of these organisms in dental hospital using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S ribosomal RNA (16S rRNA) gene and DNA gyrase subunit B (gyrB) of each species of A. baumanii complex species. Swab samples were collected from ten dental spittoon units in dental hospital, and the distribution of A. baumanii complex species was investigated using a multiplex PCR. Results: These primers were able to distinguish each species of A. baumanii complex species clearly. A. baumanii and A. calcoaceticus were detected at 20.0% and 10.0% in ten swab samples, respectively. On the other hand, A. nosocomialis, A. lowffii, and A. pittii were detected from no sample. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.
文摘Purpose: Recently, Candida haemulonii complex (Candida haemulonii, Candida duobushaemulonii and Candida haemulonii var. vulnera) and two genetically close species (Candida pseudohaemulonii and Candida auris) have emerged as an opportunistic fungal pathogen associated with various infectious diseases of humans, and most of those isolates have displayed antifungal resistance. Because it is difficult to differentiate these microorganisms by a current technique, unfortunately, it is important to establish a method for identifying them accurately. The purpose of the present study was to design species-specific primers in order to identify and detect C. auris, C. pseudohaemulonii, and C. haemulonii complex, i.e. , C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera using a multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 26S rRNA, 18S rRNA, and RPB1 genes and ITS region of five Candida species. Results: The multiplex PCR method developed in this study was able to distinguish five Candida species clearly. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and works without requiring DNA extraction.
文摘Cassava mosaic disease (CMD) caused by Cassava Mosaic Begomoviruses (CMBs) is one of the most devastating crop diseases and a major constraint for cassava production. In order to ensure surveillance for epidemic prevention, low-cost diagnostic tools are appropriate for large-scale testing of cassava viruses. Multiplex PCR diagnosis is one approach that can reduce diagnostic costs and delays. A multiplex PCR approach was developed for simultaneous detection of African cassava mosaic virus (ACMV), East African Cassava Mosaic Virus and East African cassava mosaic Cameroon virus (EACMV/CM) in Togo CMD-infected cassava leaves. Three primers pairs were used to target their respective viruses in a single tube PCR. Multiplex PCR detected ACMV, EACMV and EACMV/CM in plant DNA extracts prepared from cassava leaves infected with CMB. The primers amplified 783 bp specific to ACMV, 650 bp specific to EACMV and 560 bp specific to EACMCV/CM in both uniplex and multiplex formats. Multiplex PCR is an excellent tool for the effective control of cassava diseases. .
文摘PCRs based on different genes of Actinobacillus pleuropneumoniae have been developed for detecting and identifying A. pleuropneumoniae. Some of them could amplify positive fragments from the phylogenetically closely related species bacteria. To improve veracity and specificity of PCR, a species-specific multiplex PCR assay was developed to identify and detect A. pleuropneumoniae, based on the 3′-terminus of the species-specific apxIVA gene and the already existing species-specific primers in the omlA gene. Both 346-bp and 950-bp fragments could be simultaneously amplified from all A. pleuropneumoniae reference strains and isolates, and the species specificity of the assay was evaluated with a collection of ten strains representing eight different species bacteria including species normally found in the respiratory tracts of swine. All of these strains turned out negative in the multiplex PCR. All sequences of products of multiplex PCR randomly sampled were also correct. The sensitivity of the multiplex PCR was determined to be 10 pg of A. pleuropneumoniae DNA. The multiplex PCR and bacterial isolation were compared to determine their sensitivities by using experimentally infected pigs and clinical disease pigs. The multiplex PCR was more sensitive than bacterial isolation. The multiplex PCR was also evaluated on mixed bacterial cultures from clinical healthy pigs. 26/100 (26%) of the subclinically infected pigs were detected from clinical healthy pigs. The results indicate that the multiplex PCR assay is a sensitive, highly specific, and effective diagnostic tool for identification and detection of A. pleuropneumoniae.
文摘Objective:To evaluate microscopy,OptiMAL<sup>?</sup> and multiplex PCR for the identification of Plasmodium falciparum（P.falciparum） and Plasmodium vivax（P.vivax） from the field isolates of Bikaner,Rajasthan（Northwest India）.Methods:In this study,a multiplex PCR（P.falciparum and P.vivax） was further developed with the incorporation of Plasmodium malariae（P.malariae） specific primer and also a positive control.The performance of microscopy,plasmodium lactate dehydrogenase（pLDH） based malaria rapid diagnostic test OptiMAL<sup>?</sup> and 18S rRNA gene based multiplex PCR for the diagnosis of P.falciparum and P.vivax was compared.Results:The three species multiplex PCR if.falciparum,P.vivax and P.malariae) with an inbuilt positive control was developed and evaluated.In comparison with multiplex PCR,which showed the sensitivity and specificity of 99.36%（95%CI,98.11%-100.00%） and 100.00%（95%CI,100.00%-100.00%）,the sensitivity and specificity of microscopy was 90.44%（95%CI,88.849-95.04%） and 99.22%（95% CI,97.71%-100.00%）,and OptiMAL<sup>?</sup> was 93.58%（95%CI,89.75%-97.42%） and 97.69%（95%CI, 95.10%-100.00%）.The efficiencies were 99.65%,95.10%and 95.45%for multiplex PCK.microscopy and OptiMAL<sup>?</sup>.respectively.Conclusions:Our results raise concerns over the overall sensitivities of microscopy and OptiMAL<sup>?</sup>,when compared to the multiplex PCR and thus stress the need for new molecular interventions in the accurate detection of the malarial parasites.This further highlights the fact that further developments are needed to improve the performance of rapid diagnostic tests at field level.
基金Supported by Srinakharinwirot University(Grant No.034/2558)
文摘Objective: To develop the multiplex PCR method based on the internal transcribed spacer 2 to discriminate the intestinal trematodes, Centrocestus caninus(C. caninus), and Stellantchasmus falcatus(S. falcatus).Methods: Four species of heterophyid trematodes including C. caninus, S. falcatus,Haplorchis taichui and Haplorchoides sp. were amplified and the specific primer was designed based on the internal transcribed spacer 2 region. Two specific primers were used to validate the optimized PCR conditions: the specificity test and the sensitivity test.Results: Both of these specific primers confirmed the specificity through multiplex PCR reaction which generated both PCR products(231 and 137 bp) in the mixed DNA template of C. caninus and S. falcatus with no cross-reaction with other heterophyid trematodes. The optimum annealing temperature of both primers was 54–59℃. The sensitivity test used the two-fold serial dilution DNA template, which was concentrated between 10 and 0.312 5 ng/mL. The lowest concentration of the DNA template of this multiplex PCR was 2.5 ng/mL.Conclusions: The technique described here proved to be a species-specific technique and was found to be a rapid method for the diagnosis of C. caninus and S. falcatus in terms of the larval and adult stages in intermediate and/or definitive hosts in the endemic area.
文摘Recently, the incidence of<span> </span><i><span>Candida</span></i><span> infections has substantially increased. Conventional identification methods for </span><i><span>Candida</span></i><span> species are technically difficult to conduct and cannot accurately distinguish each species. The purpose of the present study was to design primers to identify and detect simultaneously</span><span> </span><span>eight medically important </span><i><span>Candida</span></i><span> species using one-step multiplex PCR. PCR primers were designed based on partial sequences of intergenic spacer (IGS) and internal transcribed spacer (ITS) genes of eight medically important </span><i><span>Candida</span></i><span> species. These primers were able to distinguish each </span><i><span>Candida</span></i><span> species and did not display cross-reactivity with representative </span><i><span>Candida </span></i><span>species other than the eight</span><i><span> Candida</span></i><span> species. Moreover, our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.</span>
文摘Hypertrophic cardiomyopathy (HCM) is one of the diseases damaging people health most badly and some mutations of exons in cardiac troponin I (cTnI) gene are closely associated with family hypertrophic cardiomyopathy (FHCM).A microarray was fabricated to screen mutations in exons 3,5,7,and 8 in cTnI gene.Primers were designed for the PCR (polymerase chain reaction) to amplify the target DNA fragments from fresh blood samples.In order to simplify the PCR process,multiplex PCR technology was investigated in detail.The concentration of Mg^(2+) played an important role in multiplex PCR process,a properly low concentration of Mg^(2+) submitted a better speciality of PCR products.The speciality was also favored when the annealing temperature was reasonably enhanced and 64℃is the optimal annealing temperature for the multiplex PCR systems.When applying the fabricated gene-chip to detect the target fragments from PCR mixture,the signal intensity sequence is in accordance with that from theoretic estimate.
基金supported by AESI-ISC Ⅲ grant number PI14C Ⅲ/00014
文摘Objective:To develop a new technique for diagnosis of Plasmodium knowlesi and at the same time to be able to discriminate among the diverse species of Plasmodium causing human malaria.Methods:In this study the nested multiplex malaria PCR was redesigned,targeting the 18S rR NA gene,to identify the fifth human Plasmodium species,Plasmodium knowlesi,together with the other human Plasmodium(Plasmodium falciparum,Plasmodium vivax,Plasmodium ovale and Plasmodium malariae)by amplified fragment size using only two amplification processes and including an internal reaction control to avoid false negatives.Results:The technique was validated with 91 clinical samples obtained from patients with malaria compatible symptoms.The technique showed high sensitivity(100%)and specificity(96%)when it was compared to the reference method employed for malaria diagnosis in the Instituto de Salud Carlos栿and a published real-time PCR malaria assay.Conclusions:The technique designed is an economical,sensitive and specific alternative to current diagnosis methods.Furthermore,the method might be tested in knowlesi-malaria endemic areas with a higher number of samples to confirm the quality of the method.
基金supported by the National Natural Science Foundation of China (No. 31772843)the Natural Sci- ence Foundation of Guangxi Province (No. AA17204080-4)the Fundamental Research Funds for the Central Uni- versities (No. 201762014)
文摘Iwagaki oyster,Crassostrea nippona,widely distributes along the seashore of Eastern Asia,and has been considered to be a potential breeding species due to its delicious taste and high commercial value.In order to study its genetic background and population structure,we developed 46 novel polymorphic microsatellite markers using next-generation sequencing technique and characterized them in 30 individuals.The number of alleles ranged from 3 to 22,while the observed and expected heterozygosities varied from 0.133 to 1.000 and 0.455 to 0.949,respectively.Fifteen microsatellite markers were selected and grouped into five highly informative multiplex PCRs for C.nippona.We evaluated and validated these multiplex PCRs in a cultured population including 173 candidate parents and 486 offspring.In actual parentage analysis,80%of the offspring were correctly assigned to their parental pairs using three multiplex PCRs.Furthermore,the success rate of parentage assignment reached 96%when the other two multiplex PCRs were added.These 46 microsatellite loci with high variability and the five multiplex PCRs described here provide a powerful tool for pedigree reconstruction,resource conservation and selective breeding program of C.nippona.
基金Supported by the International Cooperation Project of Heilongjiang Science and Technology Department (WC05B08)
文摘Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system,different primer ratios and annealing temperatures in SSR marker amplification.Concentration and gradient experiments for four components(enzyme,MgCl2,DNA template and dNTPs) in PCR system were used in the research with the concentration of the other component remained the same;the orthogonal design L9(34) was applied in the optimization of four sets of primers(STM0014,Pat,SSI,and UGP) in the reaction system at three levels;the temperature gradient selection was used to find out the optimum annealing temperature for the primer.The optimized multiplex PCR system of potato SSR marker with a total volume of 20 μL:2.5 μL 25 mmol · L-1 MgCl2,0.6 μL 10 mmol · L-1 dNTPs,0.8 U Taq,80 ng DNA template was ultimately established through the comparison and analysis of test results;the ratio of four pairs of 4 mmol · L-1 primers was 2:1:2:3,and the annealing temperature was 54.7℃.The optimized reaction system could be repeated stably;and the stable and reliable amplification results were able to clearly distinguish different potato varieties.This research built the solid foundation for the further study of genetic diversity of potato germplasms and construction of DNA fingerprinting..
文摘Purpose: Although the genus Candida is frequently isolated from human oral cavities, the distribution at the species level of these organisms has been little reported. The purpose of the present study was to assess the distribution at the species level of the genus Candida in human oral cavities. Methods: This study was performed using culture and Multiplex PCR methods. Moreover, the genotyping classification of C. albicans was analyzed with a PCR. Results: Of all subjects (n = 90), detection frequency of genus Candida was 42.2%. Genus Candida was not detected in the subjects between 0 to 9 years old, and there was no difference in the detection frequencies of this organism among each generation from 10s to 80s. C. albicans was the most dominant species, followed by C. parapsilosis, C. glabrata, and C. dubliniensis. Plural Candida species tended not to be detected in the individual sample. Genotype A was dominant in the C. albicans isolates. Conclusion: These results indicated that C. albicans of genotype A was dominant and that the genus Candida rarely coexists with other Candida species, in each individual oral cavity.
文摘Purpose: Fusobacterium nucleatum is an opportunistic pathogen involved in periodontal diseases, extraoral infections, and colorectal cancer. Fusobacterium necrophorum causes a variety of necrotic infections. F. nucleatum and F. necrophorum are classified into five and two subspecies, respectively. Conventional identification methods were technically hard to distinguish each subspecies of two Fusobacterium species accurately. The purpose of the present study was to design primers to identify two medically important Fusobacterium species at the subspecies level, using one-step multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S ribosomal RNA (16S rRNA) gene, RNA polymerase B (rpoB) gene, and DNA gyrase subunit B (gyrB) of each subspecies of F. nucleatum and F. necrophorum. Results: These primers were able to distinguish each subspecies of F. nucleatum and F. necrophorum and did not display cross-reactivity with representative Fusobacterium species other than F. nucleatum and F. necrophorum. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.
文摘Objective To establish multiplex PCR-based assays for detecting H.influenzae and H.parainfluenzae.And the PCR-based assays were applied to detect the carriage rates of H.influenzae and H.parainfluenzae in nasopharyngeal swab specimens which were collected from healthy children.Methods Multiplex primers for species-specific PCR were designed by using DNAstar soft based on the sequences of 16S rRNA genes from genus Haemophilus to detect H.influenzae and H.parainfluenzae.Results The sensitivity of the 16S rRNA PCR assay for detecting H.influenzae and H.parainfluenzae was 97.53% and 100% respectively,and the specificity was 95.89% and 96.63% respectively.Youden's Index on the ability to detect H.influenzae and H.parainfluenzae was 0.9342 and 0.9663 respectively.666 nasopharyngeal swab specimens were collected from healthy children.The detection rates of H.influenzae and H.parainfluenzae were 14.11% and 16.07% respectively by using isolation and culture methods.The detection rates of H.influenzae and H.parainfluenzae were 43.54% and 57.96% respectively by 16S rRNA PCR assays.The carriage rates of serotypes a,b,c,d,e,f and non-typeable isolates were 0% (0/666),0.15% (1/666),1.20% (8/666),0.15% (1/666),1.20% (8/666),1.80% (12/666),95.50% (636/666) respectively.Conclusion The multiplex PCR assays were very rapid,reliable and feasible methods for detection of H.influenzae and H.parainfluenzae in pharyngeal swab specimens which were compared to conventional isolation and culture methods.95.5% of H.influenzae strains in healthy children were nontypeable.The encapsulated or typable strains were mainly three serotypes which was c,e,and f serotype.
基金supported by grants from the Priority Project on Infectious Disease Control and Prevention(2012ZX10004215,2013ZX10004610)from Ministry of Health,China,and the Science Foundation for the State Key Laboratory for Infectious Disease Prevention and Control from China(Grant No.2015SKLID508)the National Natural Science Foundation of China(Grant No.81671985)and(Grant No.81170009)
文摘Objective Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction(PCR) and capillary electrophoresis(MPCE) assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebacterium diphtheriae, and Streptococcus pyogenes. Methods Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. Results The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10^(-7) to 10^(-2) ng/μL. Furthermore, analysis of the 152 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. Conclusion This study revealed that the MPCE assay is a rapid, reliable, and high-throughput method with high specificity and sensitivity. This assay has great potential in the molecular epidemiological survey of respiratory pathogens.
基金supported by the Shandong Seed Projectthe National Natural Science Foundation of China(No.31372524)Science and Technology Development Plan of Shandong Province,China(No.2014GHY 115002)
文摘There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.
基金National Basic Research Program of China (No. 2001CB109001)National High-Tech Research Program of China (No. 2002AA212041)
文摘Objective To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray. Methods Two sets of the multiplex PCR system were used to amplify the target genes in genetically modified (GM) soybean. Seventeen capture probes (PCR products) and 17 pairs of corresponding primers were designed according to the genetic characteristics of Rroundup Ready soybean (GTS40-3-2), maize (Mon810, Nk603, GA21), canola (T45, MS1/RF1), and rice (SCK) in many identified GM crops. All of the probes were categorized and identified as species-specific probes. One negative probe and one positive control probe were used to assess the efficiency of all reactions, and therefore eliminate any false positive and negative results. After multiplex PCR reaction, amplicons were adulterated with Cy5-dUTP and hybridized with DNA microarray. The array was then scanned to display the specific hybridization signals of target genes. The assay was applied to the analysis of sample of certified transgenic soybean (Roundup Ready GTS40-3-2) and canola (MS1/RF1). Results A combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS1/RF1 canola with a great specificity and reliability. Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events, indicating a high sensitivity. Conclusion A combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.
基金supported by a grant from the Startup Foundation for Doctors of Heilongjiang Bayi Agricultural University, China (B2009-4)
文摘Staphylococcus aureus, Escherichia coli and Bacillus cereus are the major agents of cow endometritis in dairy cows. A multiplex PCR (SEB-mPCR) was established based on the conserved genes of S. aureus, E. coli and B. cereus, and the detection limits were 103, 102 and 103 CFU mL-1, respectively. SEB-mPCR could not amplify genomic DNA of pathogenic bacteria of other common bovine diseases. A total of 309 vaginal discharge samples from cows with endometritis were tested by SEB-mPCR. Of the samples, 23.95% had the three kinds of bacteria detected, 17.15% had S. aureu and E. coli, 9.39% had E. coli and B. cereus, and 9.71% had S. aureus and B. cereus. The rates of infections with S. aureus, E. coli and B. cereus were 11.35, 16.18 and 9.06%, respectively. Therefore, SEB-mPCR has a potential as a diagnosis tool for endometritis in dairy cows.
