BACKGROUND Hepatitis C virus(HCV),hepatitis B virus(HBV),and human immunodeficiency virus 1(HIV-1)are the most epidemic blood-borne viruses,posing threats to human health and causing economic losses to nations for com...BACKGROUND Hepatitis C virus(HCV),hepatitis B virus(HBV),and human immunodeficiency virus 1(HIV-1)are the most epidemic blood-borne viruses,posing threats to human health and causing economic losses to nations for combating the infection transmission.The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive,but they are more accurate than sero-logical testing.AIM To develop a rapid,cost-effective,and accurate diagnostic multiplex polymerase chain reaction(PCR)assay for simultaneous detection of HCV,HBV,and HIV-1.METHODS The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electro-phoretic molecular weight inside each viral genome.Therefore,this diagnostic method will be appropriate for application in both conventional(combined with electrophoresis)and real-time PCR facilities.Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus.Then,Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay.RESULTS The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay.The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis.Compared to related published assays,the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays.CONCLUSION This study provides a simple,cost-effective,and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses;this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.展开更多
Library construction is a common method used to screen target genes in molecular biology.Most library constructions are not suitable for a small DNA library(<100 base pair(bp))and low RNA library output.To maximize...Library construction is a common method used to screen target genes in molecular biology.Most library constructions are not suitable for a small DNA library(<100 base pair(bp))and low RNA library output.To maximize the library’s complexity,error-prone polymerase chain reaction(PCR)was used to increase the base mutation rate.After introducing the DNA fragments into the competent cell,the library complexity could reach 109.Library mutation rate increased exponentially with the dilution and amplification of error-prone PCR.The error-prone PCR conditions were optimized including deoxyribonucleotide triphosphate(dNTP)concentration,Mn^(2+)concentration,Mg^(2+)concentration,PCR cycle number,and primer length.Then,a RNA library with high complexity can be obtained by in vitro transcription to meet most molecular biological screening requirements,and can also be used for mRNA vaccine screening.展开更多
BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in di...BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in diagnosing chromosomal structural abnormalities is limited.In this article,we report a confusing QF-PCR finding in a pregnant woman who underwent amniocentesis.CASE SUMMARY The short tandem repeat marker AMXY(Xp22.2/Yp11.2)located on the sex chromosome exhibited a trisomic biallelic pattern,indicating that the karyotype of the fetus might be 47,XYY.Chromosome analysis performed on cultured amniocytes showed a normal male karyotype of the fetus.Copy number variation sequencing confirmed a 500 kb duplication at Yp11.2-Yp11.2(chrY:6610001_7110000)and a 250 kb duplication at Yp11.2-Yp11.2(chrY:7110001_7360000).CONCLUSION In conclusion,the comprehensive application of different methods could achieve a higher detection rate and accuracy for the prenatal diagnosis of chromosomal disorders through chromosomal testing.展开更多
Introduction: Transitioning to more efficacious Antiretrovirals for HIV infected pregnant women and infant prophylaxis has reduced Mother to child transmission of HIV significantly. This study aimed to determine HIV i...Introduction: Transitioning to more efficacious Antiretrovirals for HIV infected pregnant women and infant prophylaxis has reduced Mother to child transmission of HIV significantly. This study aimed to determine HIV infection status in HIVexposed infants who had their first DNA polymerase chain reaction test in our molecular Laboratory. Subjects, Materials and Methods: Dried Blood Spots for HIV DNA results from 5 states between 2009 and 2020 were analyzed in the PCR laboratory of the Federal Teaching Hospital, Gombe. Results: Nine thousand eight hundred and twenty-three Human Immunodeficiency Virus Deoxyribonucleic acid polymerase Chain Reaction results were analysed;4937 (50.2%) were males. During the study period, there was an overall declining trend in the mother-to-child transmission rate from 3.8% in 2009 to 1.0% in 2020. 6120 (62.3%) of HIV + mothers received Highly active antiretroviral therapy HAART before pregnancy. 7845 (76.2%) of the infants received Nevirapine prophylaxis. Dried blood spot samples were collected from 4077 (41.5%) at 6 - 8 weeks. 8438 (85.9%) received cotrimoxazole. 9469 (96.4%) were ever breastfed. Of the 9823 HIV DNA PCR results, 255 (2.6%) were positive while 69/4077 (1.7%) and 109/2662 (4.1%) were positive for HIV DNA at 6 - 8 weeks and > 12 weeks respectively. (p = 0.001). 86/747 (11.5%) of infants whose HIV-positive mothers received no ARVS were HIV DNA positive. (p = 0.001). 106/884 (12.0%) of infants who had no Antiretroviral prophylaxis had positive HIV DNA results;7/413 (1.7%) with Zidovudine/Nevirapine prophylaxis had positive results. (p = 0.001). 246/9469 (2.6%) of infants that were ever breastfed were positive for HIV DNA;11/354 (3.0%) that never breastfed had positive HIV DNA. Conclusion: Lack of maternal/infant ARVs and prolonged breastfeeding increased the risk of infant HIV infection.展开更多
BACKGROUND Helicobacter pylori(H.pylori)infection is closely associated with the etiology of a variety of gastric diseases.The effective eradication of H.pylori infection has been shown to reduce the incidence of gast...BACKGROUND Helicobacter pylori(H.pylori)infection is closely associated with the etiology of a variety of gastric diseases.The effective eradication of H.pylori infection has been shown to reduce the incidence of gastric carcinoma.However,the rate of H.pylori eradication has significantly declined due to its increasing resistance to antibiotics,especially to clarithromycin.Therefore,the detection of clarithromycin resistance is necessary prior to the treatment of H.pylori.Although many studies have been conducted on the use of polymerase chain reaction(PCR)-based tests to detect clarithromycin resistance in stool samples,no accurate data on the feasibility of these tests are available.Here,we performed a meta-analysis to assess the feasibility of these noninvasive tests.AIM To evaluate the reliability of PCR-based tests for detecting H.pylori clarithromycin resistance in stool samples.METHODS We searched PubMed,Medline,Embase,and other databases for articles that evaluated the value of the PCR analysis of stool samples for detecting the resistance of H.pylori to clarithromycin.We collected cross-sectional studies that met the inclusion criteria.Diagnostic accuracy measures were pooled using a random-effects model.The risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool.Subgroup analysis was also conducted according to PCR type,purification technique,reference standard,mutation site,sample weight,number of patients,and age group,and the clinical utility of diagnostic tests was evaluated using the Likelihood Ratio Scatter Graph.RESULTS Out of the 1818 identified studies,only 11 met the eligibility criteria,with a total of 592 patients assessed.A meta-analysis of the random-effect model showed that PCR-based analysis of stool samples had high diagnostic accuracy for detecting clarithromycin resistance in patients infected with H.pylori.The combined sensitivity was 0.91[95%confidence interval(CI):0.83-0.95],Q=30.34,and I2=67.04,and the combined specificity was 0.97(95%CI:0.62-1.00),Q=279.54,and I2=96.42.The likelihood ratio for a positive test was 33.25(95%CI:1.69-652.77),and that for a negative test was 0.10(95%CI:0.05-0.18),with an area under the curve of 0.94.The diagnostic odds ratio was 347.68(95%CI:17.29-6991.26).There was significant statistical heterogeneity,and the sub-analyses showed significant differences in the number of patients,sample weight,purification methods,PCR types,mutation points,and reference standards.The included studies showed no risk of publication bias.CONCLUSION PCR-based tests on stool samples have high diagnostic accuracy for detecting H.pylori clarithromycin resistance.展开更多
BACKGROUND Recently,stool multiplex polymerase chain reaction(PCR)tests have been developed for identifying diarrhea-causing bacterial pathogens.Furthermore,fecal calprotectin is a well-known effective marker for inte...BACKGROUND Recently,stool multiplex polymerase chain reaction(PCR)tests have been developed for identifying diarrhea-causing bacterial pathogens.Furthermore,fecal calprotectin is a well-known effective marker for intestinal mucosal inflammation.AIM To evaluate the efficacy of stool multiplex PCR and fecal calprotectin in acute infectious diarrhea.METHODS Overall,400 patients with acute infectious diarrhea were enrolled from Kangdong Sacred Heart Hospital(January 2016 to December 2018).Multiplex PCR detected 7 enteropathogenic bacteria including Salmonella,Campylobacter,Shigella,Escherichia coli O157:H7,Aeromonas,Vibrio,and Clostridium difficile.We reviewed clinical and laboratory findings using stool multiplex PCR.RESULTS Stool multiplex PCR test detected considerably more bacterial pathogens than stool culture(49.2%vs 5.2%),with Campylobacter as the most common pathogen(54%).Patients with positive stool PCR showed elevated fecal calprotectin expression compared to patients with negative stool PCR(1124.5±816.9 mg/kg vs 609±713.2 mg/kg,P=0.001).C-reactive protein(OR=1.01,95%CI:1.001-1.027,P=0.034)and sigmoidoscopy-detected colitis(OR=4.76,95%CI:1.101-20.551,P=0.037)were independent factors in stool PCR-based detection of bacterial pathogens.Sensitivity and specificity of calprotectin were evaluated to be 70.5%and 60.9%,respectively(adjusted cut-off value=388 mg/kg).CONCLUSION Stool multiplex PCR test has increased sensitivity in detecting pathogens than conventional culture,and it is correlated with calprotectin expression.Stool multiplex PCR and calprotectin may be effective in predicting clinical severity of infectious diarrhea.展开更多
AIM: To design, optimize and validate a rapid,internally controlled real-time polymerase chain reaction(RT-PCR) test for herpes simplex virus(HSV) in the diagnosis of necrotizing herpes stromal keratitis.· METHOD...AIM: To design, optimize and validate a rapid,internally controlled real-time polymerase chain reaction(RT-PCR) test for herpes simplex virus(HSV) in the diagnosis of necrotizing herpes stromal keratitis.· METHODS: Tears alone or together with corneal epithelium scrapings from 30 patients(30 eyes)suspected of necrotizing herpes stromal keratitis were tested for HSV DNA by RT-PCR. The samples were collected during the first visit and then on the subsequent 7, 14, 28, 42, and 56 d. The symptoms of the patients were scored before treatment to determine the correlation between HSV concentration in the corneal epithelium scrapings and clinical scores.·RESULTS: The positive rate(46.4%) in the corneal epithelium group before the therapy was significantly higher than that(13.3%) in the tears group(P =0.006).There were 13 positive HSV patients before the therapy,the concentration of HSV DNA in corneal epithelium scrapings group was significantly higher than that in the tears group(paired t-test, P =0.0397). Multilevel mixedeffects model analysis showed that the difference between the corneal epithelium scrapings group and the tears group was statistically significant(P =0.0049). The Spearman rank correlation analysis indicated a positive correlation between the HSV concentration in the corneal epithelium scrapings and clinical scores before the treatment(r =0.844, P <0.0001).· CONCLUSION: RT-PCR appears to be a powerful molecular tool for the diagnosis of necrotizing herpes stromal keratitis.展开更多
BACKGROUND Gastric cancer(GC)is one of the most prevalent malignant tumors that endangers human health.Early diagnosis is essential for improving the prognosis and survival rate of GC patients.Ring finger protein 180(...BACKGROUND Gastric cancer(GC)is one of the most prevalent malignant tumors that endangers human health.Early diagnosis is essential for improving the prognosis and survival rate of GC patients.Ring finger protein 180(RNF180)is involved in the regulation of cell differentiation,proliferation,apoptosis,and tumorigenesis,and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC.Thus,methylated RNF180 can be used as a potential biomarker for GC diagnosis.AIM To use droplet digital polymerase chain reaction(ddPCR)to quantify the methylation level of the RN180 gene.A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.METHODS The primer and probe were designed and selected,the conversion time of bisulfite was optimized,the ddPCR system was adjusted by primer concentration,amplification temperature and amplification cycles,and the detection limit of ddPCR was determined.RESULTS The best conversion time for blood DNA was 2 h 10 min,and that for plasma DNA was 2 h 10 min and 2 h 30 min.The results of ddPCR were better when the amplification temperature was 56°C and the number of amplification cycles was 50.Primer concentrations showed little effect on the assay outcome.Therefore,the primer concentration could be adjusted according to the reaction system and DNA input.The assay required at least 0.1 ng of input DNA.CONCLUSION In summary,a ddPCR assay was established to detect methylated RNF180,which is expected to be a new diagnostic biomarker for GC.展开更多
According to the fact that the abnormalities of visual pigment genes were always involved in the changing of the exon 5, two oligonucleotide primers were designed to amplify the exon 5 of red pigment gene and green pi...According to the fact that the abnormalities of visual pigment genes were always involved in the changing of the exon 5, two oligonucleotide primers were designed to amplify the exon 5 of red pigment gene and green pigment gene. After electrophoresis of the PCR products digested with Rsal or Sau3A, the DNA fragments from the exon 5 of red pigment gene (RPG) and green pigment gene (GPG) were separated since there are different restriction endonuclease sites. On the other hand, we analyzed the exon 5 rela...展开更多
We report a novel method for rapid,colorimetric detection of a specific deoxyribonucleic acid(DNA)sequence by carrying out a polymerase chain reaction in the presence of gold nanoparticles functionalized with two prim...We report a novel method for rapid,colorimetric detection of a specific deoxyribonucleic acid(DNA)sequence by carrying out a polymerase chain reaction in the presence of gold nanoparticles functionalized with two primers.Extension of the primers when the target DNA is present as a template during the polymerase chain reaction process affords the complementary sequences on the gold nanoparticle surfaces and results in the formation of gold nanoparticle aggregates with a concomitant color change from red to pinkish/purple.This method provides a convenient and straightforward solution for ultrasensitive DNA detection without any further post-treatment of the polymerase chain reaction products being necessary,and is a promising tool for rapid disease diagnostics and gene sequencing.展开更多
Deoxyribonucleic acid(DNA)hydrogels,a three-dimensional(3 D)network made from DNA chains,have attracted great attention because of its molecular programmability,excellent biocompatibility and wide biomedical applicati...Deoxyribonucleic acid(DNA)hydrogels,a three-dimensional(3 D)network made from DNA chains,have attracted great attention because of its molecular programmability,excellent biocompatibility and wide biomedical applications.Construction of hydrogel incorporating genetic function is still a challenge because of the limitations in available preparation methods.Herein,we develop a polymerase chain reaction(PCR)based strategy to construct gene integrated hydrogel to mimic the biofunction of nucleus zone.DNA primers were chemically modified by methacrylamide,which were used as modular primers in PCR to hybridize with template plasmid DNA,yielding methacrylamide functionalized gene(Acry-gene).Afterwards,Acry-gene was chemically cross-linked and compressed via free radical polymerization of terminal group methacrylamide to form a threedimensional gene network,namely gene hydrogel.The gene hydrogel retained the genetic function and expressed protein successfully in a cell free protein expression system.This work provides a general approach for the construction of biofunctional gene hydrogel which mimics bioprocesses,showing great potential in biomedicine and biomimetic fields.展开更多
Leptospirosis,a major zoonotic disease widespread in the world,would infect humans and other animals by direct contacting with the soil or water contaminated by the pathogenic leptospires.This report analyzes the accu...Leptospirosis,a major zoonotic disease widespread in the world,would infect humans and other animals by direct contacting with the soil or water contaminated by the pathogenic leptospires.This report analyzes the accuracy of polymerase chain reaction(PCR)assay used in the detection of leptospira specific genes in humans.Seventeen published articles which included 2526 samples satisfied all inclusion standards and were applied to meta-analysis.Pooled sensitivity and specificity were 0.73(95%CI 0.70-0.76)and 0.94(95%CI 0.93-0.95),respectively.Heterogeneity was statistically significant among studies,but none of the sources for heterogeneity(disease stage,PCR type,targeted genes)could adequately interpret this finding.Meta-regression advised that real-time PCR(qPCR)targeted 16 s rRNA would be the best choice for early detection of cases during the acute stage of the disease and was accounted as a good screening tool in all stages of the disease.展开更多
Among different endangered animal species,snakes are the most neglected creature looked at with apathy and therefore,are ruthlessly killed,illegally trafficked,and poached for their venom,lucrative skin,meat,and bones...Among different endangered animal species,snakes are the most neglected creature looked at with apathy and therefore,are ruthlessly killed,illegally trafficked,and poached for their venom,lucrative skin,meat,and bones for manufacturing of medicines,accessories,and food items.Establishing the identity of the endangered snake species is important for punishing the offenders under Wildlife Protection Act(WPA)(1972)but morphological characters fail to establish identity as they are often altered.The technique of identification of snake species at molecular level holds very effective conclusion in punishing offender.Here,we have constructed and demonstrated a novel multiplexing polymerase chain reaction technique,using 16S rRNA and C-mos gene for identification of four Indian snake species,namely Ptyas mucosa,Daboia russellii,Naja naja,and Xenochrophis piscator.They are listed in Appendix-II and III of convention on international trade in endangered species of wild fauna and flora and Schedule II;Part II of Indian WPA,1972.Therefore,it may be considered a functional tool for establishing species-specific identity of four Indian snake species and promising to be useful for their conservation.展开更多
The genomic DNA is the organism’s various physiological and pathological characteristics of the material base.Single nucleotide polymorphisms(single nucleotide polymorphisms,SNPs) is the genome of the most common for...The genomic DNA is the organism’s various physiological and pathological characteristics of the material base.Single nucleotide polymorphisms(single nucleotide polymorphisms,SNPs) is the genome of the most common form of genetic variation in the human genome widespread.With the completion of the Human Genome Project to stimulate,there is growing focus on research developed in this latest 3rd generation of genetic markers of practical significance.This paper describes the classification of SNPs and characteristics of the human genome SNP s research and development and the present in today’s practice,the application of design experimts to explore the direct sequencing of amplified using PCR-based method showed that SNPs.展开更多
AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl trans...AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.展开更多
Given the lack of defining features in the clinical manifestations and radiographic findings for children with mycoplasma pneumoniae pneumonia(MPP),quantitative polymerase chain reaction(qPCR)has become a useful diagn...Given the lack of defining features in the clinical manifestations and radiographic findings for children with mycoplasma pneumoniae pneumonia(MPP),quantitative polymerase chain reaction(qPCR)has become a useful diagnostic method.This study was performed to explore the relationship between the qPCR findings,clinical symptoms,and inflammatory markers in children with MPP.Four hundred children with MPP have been enrolled in this retrospective analysis.All clinical and analytical information,including mycoplasma pneumoniae(MP)PCR results,has been collected.Based on the PCR results,the patients were divided into groups with load values(copy number)<105(54 cases),2105 and<106(71 cases),2106 and<107(112 cases),>107 and<108(114 cases),and>108(49 cases).The clinical features(including symptoms and signs)and inflammatory indicators were compared among the groups.The incidence of high fever(above 39℃),thermal peak during the entire hospitalization period,fever duration,days of hospitalization,and plasma lactate dehydrogenase(LDH)levels were statistically correlated with the MP PCR load value in children with MPP.The analysis of relevance degree showed the correlative order as a thermal peak of hospitalization>duration of fever>period of hospitalization>LDH value>C-reactive protein value.The host immune response was significantly greater in the complication group than in the non-complication group.展开更多
BACKGROUND The Omicron variant of severe acute respiratory syndrome coronavirus 2(SARSCoV-2)mainly infects the upper respiratory tract.This study aimed to determine whether the probability of pulmonary infection and t...BACKGROUND The Omicron variant of severe acute respiratory syndrome coronavirus 2(SARSCoV-2)mainly infects the upper respiratory tract.This study aimed to determine whether the probability of pulmonary infection and the cycle threshold(Ct)measured using the fluorescent polymerase chain reaction(PCR)method were related to pulmonary infections diagnosed via computed tomography(CT).AIM To analyze the chest CT signs of SARS-CoV-2 Omicron variant infections with different Ct values,as determined via PCR.METHODS The chest CT images and PCR Ct values of 331 patients with SARS-CoV-2Omicron variant infections were retrospectively collected and categorized into low(<25),medium(25.00-34.99),and high(≥35)Ct groups.The characteristics of chest CT images in each group were statistically analyzed.RESULTS The PCR Ct values ranged from 13.36 to 39.81,with 99 patients in the low,155 in the medium,and 77 in the high Ct groups.Six abnormal chest CT signs were detected,namely,focal infection,patchy consolidation shadows,patchy groundglass shadows,mixed consolidation ground-glass shadows,subpleural interstitial changes,and pleural changes.Focal infections were less frequent in the low Ct group than in the medium and high Ct groups;these infections were the most common sign in the medium and high Ct groups.Patchy consolidation shadows and pleural changes were more frequent in the low Ct group than in the other two groups.The number of patients with two or more signs was greater in the low Ct group than in the medium and high Ct groups.CONCLUSION The chest CT signs of patients with pulmonary infection caused by the Omicron variants of SARSCoV-2 varied depending on the Ct values.Identification of the characteristics of Omicron variant infection can help subsequent planning of clinical treatment.展开更多
文摘BACKGROUND Hepatitis C virus(HCV),hepatitis B virus(HBV),and human immunodeficiency virus 1(HIV-1)are the most epidemic blood-borne viruses,posing threats to human health and causing economic losses to nations for combating the infection transmission.The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive,but they are more accurate than sero-logical testing.AIM To develop a rapid,cost-effective,and accurate diagnostic multiplex polymerase chain reaction(PCR)assay for simultaneous detection of HCV,HBV,and HIV-1.METHODS The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electro-phoretic molecular weight inside each viral genome.Therefore,this diagnostic method will be appropriate for application in both conventional(combined with electrophoresis)and real-time PCR facilities.Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus.Then,Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay.RESULTS The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay.The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis.Compared to related published assays,the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays.CONCLUSION This study provides a simple,cost-effective,and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses;this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.
基金Shanghai Science and Technology Commission’s“Belt and Road Initiative”International Cooperation Project,China(No.19410741800)。
文摘Library construction is a common method used to screen target genes in molecular biology.Most library constructions are not suitable for a small DNA library(<100 base pair(bp))and low RNA library output.To maximize the library’s complexity,error-prone polymerase chain reaction(PCR)was used to increase the base mutation rate.After introducing the DNA fragments into the competent cell,the library complexity could reach 109.Library mutation rate increased exponentially with the dilution and amplification of error-prone PCR.The error-prone PCR conditions were optimized including deoxyribonucleotide triphosphate(dNTP)concentration,Mn^(2+)concentration,Mg^(2+)concentration,PCR cycle number,and primer length.Then,a RNA library with high complexity can be obtained by in vitro transcription to meet most molecular biological screening requirements,and can also be used for mRNA vaccine screening.
文摘BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in diagnosing chromosomal structural abnormalities is limited.In this article,we report a confusing QF-PCR finding in a pregnant woman who underwent amniocentesis.CASE SUMMARY The short tandem repeat marker AMXY(Xp22.2/Yp11.2)located on the sex chromosome exhibited a trisomic biallelic pattern,indicating that the karyotype of the fetus might be 47,XYY.Chromosome analysis performed on cultured amniocytes showed a normal male karyotype of the fetus.Copy number variation sequencing confirmed a 500 kb duplication at Yp11.2-Yp11.2(chrY:6610001_7110000)and a 250 kb duplication at Yp11.2-Yp11.2(chrY:7110001_7360000).CONCLUSION In conclusion,the comprehensive application of different methods could achieve a higher detection rate and accuracy for the prenatal diagnosis of chromosomal disorders through chromosomal testing.
文摘Introduction: Transitioning to more efficacious Antiretrovirals for HIV infected pregnant women and infant prophylaxis has reduced Mother to child transmission of HIV significantly. This study aimed to determine HIV infection status in HIVexposed infants who had their first DNA polymerase chain reaction test in our molecular Laboratory. Subjects, Materials and Methods: Dried Blood Spots for HIV DNA results from 5 states between 2009 and 2020 were analyzed in the PCR laboratory of the Federal Teaching Hospital, Gombe. Results: Nine thousand eight hundred and twenty-three Human Immunodeficiency Virus Deoxyribonucleic acid polymerase Chain Reaction results were analysed;4937 (50.2%) were males. During the study period, there was an overall declining trend in the mother-to-child transmission rate from 3.8% in 2009 to 1.0% in 2020. 6120 (62.3%) of HIV + mothers received Highly active antiretroviral therapy HAART before pregnancy. 7845 (76.2%) of the infants received Nevirapine prophylaxis. Dried blood spot samples were collected from 4077 (41.5%) at 6 - 8 weeks. 8438 (85.9%) received cotrimoxazole. 9469 (96.4%) were ever breastfed. Of the 9823 HIV DNA PCR results, 255 (2.6%) were positive while 69/4077 (1.7%) and 109/2662 (4.1%) were positive for HIV DNA at 6 - 8 weeks and > 12 weeks respectively. (p = 0.001). 86/747 (11.5%) of infants whose HIV-positive mothers received no ARVS were HIV DNA positive. (p = 0.001). 106/884 (12.0%) of infants who had no Antiretroviral prophylaxis had positive HIV DNA results;7/413 (1.7%) with Zidovudine/Nevirapine prophylaxis had positive results. (p = 0.001). 246/9469 (2.6%) of infants that were ever breastfed were positive for HIV DNA;11/354 (3.0%) that never breastfed had positive HIV DNA. Conclusion: Lack of maternal/infant ARVs and prolonged breastfeeding increased the risk of infant HIV infection.
基金“New Xiangya Talent Projects”of The Third Xiangya Hospital of Central South University,No.JY201710.
文摘BACKGROUND Helicobacter pylori(H.pylori)infection is closely associated with the etiology of a variety of gastric diseases.The effective eradication of H.pylori infection has been shown to reduce the incidence of gastric carcinoma.However,the rate of H.pylori eradication has significantly declined due to its increasing resistance to antibiotics,especially to clarithromycin.Therefore,the detection of clarithromycin resistance is necessary prior to the treatment of H.pylori.Although many studies have been conducted on the use of polymerase chain reaction(PCR)-based tests to detect clarithromycin resistance in stool samples,no accurate data on the feasibility of these tests are available.Here,we performed a meta-analysis to assess the feasibility of these noninvasive tests.AIM To evaluate the reliability of PCR-based tests for detecting H.pylori clarithromycin resistance in stool samples.METHODS We searched PubMed,Medline,Embase,and other databases for articles that evaluated the value of the PCR analysis of stool samples for detecting the resistance of H.pylori to clarithromycin.We collected cross-sectional studies that met the inclusion criteria.Diagnostic accuracy measures were pooled using a random-effects model.The risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool.Subgroup analysis was also conducted according to PCR type,purification technique,reference standard,mutation site,sample weight,number of patients,and age group,and the clinical utility of diagnostic tests was evaluated using the Likelihood Ratio Scatter Graph.RESULTS Out of the 1818 identified studies,only 11 met the eligibility criteria,with a total of 592 patients assessed.A meta-analysis of the random-effect model showed that PCR-based analysis of stool samples had high diagnostic accuracy for detecting clarithromycin resistance in patients infected with H.pylori.The combined sensitivity was 0.91[95%confidence interval(CI):0.83-0.95],Q=30.34,and I2=67.04,and the combined specificity was 0.97(95%CI:0.62-1.00),Q=279.54,and I2=96.42.The likelihood ratio for a positive test was 33.25(95%CI:1.69-652.77),and that for a negative test was 0.10(95%CI:0.05-0.18),with an area under the curve of 0.94.The diagnostic odds ratio was 347.68(95%CI:17.29-6991.26).There was significant statistical heterogeneity,and the sub-analyses showed significant differences in the number of patients,sample weight,purification methods,PCR types,mutation points,and reference standards.The included studies showed no risk of publication bias.CONCLUSION PCR-based tests on stool samples have high diagnostic accuracy for detecting H.pylori clarithromycin resistance.
文摘BACKGROUND Recently,stool multiplex polymerase chain reaction(PCR)tests have been developed for identifying diarrhea-causing bacterial pathogens.Furthermore,fecal calprotectin is a well-known effective marker for intestinal mucosal inflammation.AIM To evaluate the efficacy of stool multiplex PCR and fecal calprotectin in acute infectious diarrhea.METHODS Overall,400 patients with acute infectious diarrhea were enrolled from Kangdong Sacred Heart Hospital(January 2016 to December 2018).Multiplex PCR detected 7 enteropathogenic bacteria including Salmonella,Campylobacter,Shigella,Escherichia coli O157:H7,Aeromonas,Vibrio,and Clostridium difficile.We reviewed clinical and laboratory findings using stool multiplex PCR.RESULTS Stool multiplex PCR test detected considerably more bacterial pathogens than stool culture(49.2%vs 5.2%),with Campylobacter as the most common pathogen(54%).Patients with positive stool PCR showed elevated fecal calprotectin expression compared to patients with negative stool PCR(1124.5±816.9 mg/kg vs 609±713.2 mg/kg,P=0.001).C-reactive protein(OR=1.01,95%CI:1.001-1.027,P=0.034)and sigmoidoscopy-detected colitis(OR=4.76,95%CI:1.101-20.551,P=0.037)were independent factors in stool PCR-based detection of bacterial pathogens.Sensitivity and specificity of calprotectin were evaluated to be 70.5%and 60.9%,respectively(adjusted cut-off value=388 mg/kg).CONCLUSION Stool multiplex PCR test has increased sensitivity in detecting pathogens than conventional culture,and it is correlated with calprotectin expression.Stool multiplex PCR and calprotectin may be effective in predicting clinical severity of infectious diarrhea.
文摘AIM: To design, optimize and validate a rapid,internally controlled real-time polymerase chain reaction(RT-PCR) test for herpes simplex virus(HSV) in the diagnosis of necrotizing herpes stromal keratitis.· METHODS: Tears alone or together with corneal epithelium scrapings from 30 patients(30 eyes)suspected of necrotizing herpes stromal keratitis were tested for HSV DNA by RT-PCR. The samples were collected during the first visit and then on the subsequent 7, 14, 28, 42, and 56 d. The symptoms of the patients were scored before treatment to determine the correlation between HSV concentration in the corneal epithelium scrapings and clinical scores.·RESULTS: The positive rate(46.4%) in the corneal epithelium group before the therapy was significantly higher than that(13.3%) in the tears group(P =0.006).There were 13 positive HSV patients before the therapy,the concentration of HSV DNA in corneal epithelium scrapings group was significantly higher than that in the tears group(paired t-test, P =0.0397). Multilevel mixedeffects model analysis showed that the difference between the corneal epithelium scrapings group and the tears group was statistically significant(P =0.0049). The Spearman rank correlation analysis indicated a positive correlation between the HSV concentration in the corneal epithelium scrapings and clinical scores before the treatment(r =0.844, P <0.0001).· CONCLUSION: RT-PCR appears to be a powerful molecular tool for the diagnosis of necrotizing herpes stromal keratitis.
基金Supported by the National Key Research and Development Program of China,No.2020YFC2002700the National Natural Science Foundation of China,No.81972010+1 种基金the CAMS Initiative for Innovative Medicine,No.2016-I2M-1-007the Science Developing Funds of Navy General Hospital,No.CXPY201810.
文摘BACKGROUND Gastric cancer(GC)is one of the most prevalent malignant tumors that endangers human health.Early diagnosis is essential for improving the prognosis and survival rate of GC patients.Ring finger protein 180(RNF180)is involved in the regulation of cell differentiation,proliferation,apoptosis,and tumorigenesis,and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC.Thus,methylated RNF180 can be used as a potential biomarker for GC diagnosis.AIM To use droplet digital polymerase chain reaction(ddPCR)to quantify the methylation level of the RN180 gene.A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.METHODS The primer and probe were designed and selected,the conversion time of bisulfite was optimized,the ddPCR system was adjusted by primer concentration,amplification temperature and amplification cycles,and the detection limit of ddPCR was determined.RESULTS The best conversion time for blood DNA was 2 h 10 min,and that for plasma DNA was 2 h 10 min and 2 h 30 min.The results of ddPCR were better when the amplification temperature was 56°C and the number of amplification cycles was 50.Primer concentrations showed little effect on the assay outcome.Therefore,the primer concentration could be adjusted according to the reaction system and DNA input.The assay required at least 0.1 ng of input DNA.CONCLUSION In summary,a ddPCR assay was established to detect methylated RNF180,which is expected to be a new diagnostic biomarker for GC.
文摘According to the fact that the abnormalities of visual pigment genes were always involved in the changing of the exon 5, two oligonucleotide primers were designed to amplify the exon 5 of red pigment gene and green pigment gene. After electrophoresis of the PCR products digested with Rsal or Sau3A, the DNA fragments from the exon 5 of red pigment gene (RPG) and green pigment gene (GPG) were separated since there are different restriction endonuclease sites. On the other hand, we analyzed the exon 5 rela...
基金This work was supported by the“Bairen Jihua”program of Chinese Academy of Sciences,the Chinese Academy of Sciences/State Administration of Foreign Experts Affairs(CAS/SAFEA)International Partnership Program for Creative Research Teams and Suzhou Bureau of Science and Technology.
文摘We report a novel method for rapid,colorimetric detection of a specific deoxyribonucleic acid(DNA)sequence by carrying out a polymerase chain reaction in the presence of gold nanoparticles functionalized with two primers.Extension of the primers when the target DNA is present as a template during the polymerase chain reaction process affords the complementary sequences on the gold nanoparticle surfaces and results in the formation of gold nanoparticle aggregates with a concomitant color change from red to pinkish/purple.This method provides a convenient and straightforward solution for ultrasensitive DNA detection without any further post-treatment of the polymerase chain reaction products being necessary,and is a promising tool for rapid disease diagnostics and gene sequencing.
基金supported by the National Natural Science Foundation of China(21621004,21575101,21622404)
文摘Deoxyribonucleic acid(DNA)hydrogels,a three-dimensional(3 D)network made from DNA chains,have attracted great attention because of its molecular programmability,excellent biocompatibility and wide biomedical applications.Construction of hydrogel incorporating genetic function is still a challenge because of the limitations in available preparation methods.Herein,we develop a polymerase chain reaction(PCR)based strategy to construct gene integrated hydrogel to mimic the biofunction of nucleus zone.DNA primers were chemically modified by methacrylamide,which were used as modular primers in PCR to hybridize with template plasmid DNA,yielding methacrylamide functionalized gene(Acry-gene).Afterwards,Acry-gene was chemically cross-linked and compressed via free radical polymerization of terminal group methacrylamide to form a threedimensional gene network,namely gene hydrogel.The gene hydrogel retained the genetic function and expressed protein successfully in a cell free protein expression system.This work provides a general approach for the construction of biofunctional gene hydrogel which mimics bioprocesses,showing great potential in biomedicine and biomimetic fields.
基金the National Natural Science Foundation of China(81271781)the Natural Science Foundation of Zhejiang Province(LY17H190002)the Higher Education Institutions of Gansu Province of China(2019B-230)。
文摘Leptospirosis,a major zoonotic disease widespread in the world,would infect humans and other animals by direct contacting with the soil or water contaminated by the pathogenic leptospires.This report analyzes the accuracy of polymerase chain reaction(PCR)assay used in the detection of leptospira specific genes in humans.Seventeen published articles which included 2526 samples satisfied all inclusion standards and were applied to meta-analysis.Pooled sensitivity and specificity were 0.73(95%CI 0.70-0.76)and 0.94(95%CI 0.93-0.95),respectively.Heterogeneity was statistically significant among studies,but none of the sources for heterogeneity(disease stage,PCR type,targeted genes)could adequately interpret this finding.Meta-regression advised that real-time PCR(qPCR)targeted 16 s rRNA would be the best choice for early detection of cases during the acute stage of the disease and was accounted as a good screening tool in all stages of the disease.
文摘Among different endangered animal species,snakes are the most neglected creature looked at with apathy and therefore,are ruthlessly killed,illegally trafficked,and poached for their venom,lucrative skin,meat,and bones for manufacturing of medicines,accessories,and food items.Establishing the identity of the endangered snake species is important for punishing the offenders under Wildlife Protection Act(WPA)(1972)but morphological characters fail to establish identity as they are often altered.The technique of identification of snake species at molecular level holds very effective conclusion in punishing offender.Here,we have constructed and demonstrated a novel multiplexing polymerase chain reaction technique,using 16S rRNA and C-mos gene for identification of four Indian snake species,namely Ptyas mucosa,Daboia russellii,Naja naja,and Xenochrophis piscator.They are listed in Appendix-II and III of convention on international trade in endangered species of wild fauna and flora and Schedule II;Part II of Indian WPA,1972.Therefore,it may be considered a functional tool for establishing species-specific identity of four Indian snake species and promising to be useful for their conservation.
文摘The genomic DNA is the organism’s various physiological and pathological characteristics of the material base.Single nucleotide polymorphisms(single nucleotide polymorphisms,SNPs) is the genome of the most common form of genetic variation in the human genome widespread.With the completion of the Human Genome Project to stimulate,there is growing focus on research developed in this latest 3rd generation of genetic markers of practical significance.This paper describes the classification of SNPs and characteristics of the human genome SNP s research and development and the present in today’s practice,the application of design experimts to explore the direct sequencing of amplified using PCR-based method showed that SNPs.
基金Supported by grant from Fundamental Research Grant Scheme by Ministry of Higher Education(MoHE)600-IRMI/FRGS 5/3(101/2019).
文摘AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.
基金This study was supported by the Chongqing Science and Health Joint Medical Research Project(No.8187011078)。
文摘Given the lack of defining features in the clinical manifestations and radiographic findings for children with mycoplasma pneumoniae pneumonia(MPP),quantitative polymerase chain reaction(qPCR)has become a useful diagnostic method.This study was performed to explore the relationship between the qPCR findings,clinical symptoms,and inflammatory markers in children with MPP.Four hundred children with MPP have been enrolled in this retrospective analysis.All clinical and analytical information,including mycoplasma pneumoniae(MP)PCR results,has been collected.Based on the PCR results,the patients were divided into groups with load values(copy number)<105(54 cases),2105 and<106(71 cases),2106 and<107(112 cases),>107 and<108(114 cases),and>108(49 cases).The clinical features(including symptoms and signs)and inflammatory indicators were compared among the groups.The incidence of high fever(above 39℃),thermal peak during the entire hospitalization period,fever duration,days of hospitalization,and plasma lactate dehydrogenase(LDH)levels were statistically correlated with the MP PCR load value in children with MPP.The analysis of relevance degree showed the correlative order as a thermal peak of hospitalization>duration of fever>period of hospitalization>LDH value>C-reactive protein value.The host immune response was significantly greater in the complication group than in the non-complication group.
文摘BACKGROUND The Omicron variant of severe acute respiratory syndrome coronavirus 2(SARSCoV-2)mainly infects the upper respiratory tract.This study aimed to determine whether the probability of pulmonary infection and the cycle threshold(Ct)measured using the fluorescent polymerase chain reaction(PCR)method were related to pulmonary infections diagnosed via computed tomography(CT).AIM To analyze the chest CT signs of SARS-CoV-2 Omicron variant infections with different Ct values,as determined via PCR.METHODS The chest CT images and PCR Ct values of 331 patients with SARS-CoV-2Omicron variant infections were retrospectively collected and categorized into low(<25),medium(25.00-34.99),and high(≥35)Ct groups.The characteristics of chest CT images in each group were statistically analyzed.RESULTS The PCR Ct values ranged from 13.36 to 39.81,with 99 patients in the low,155 in the medium,and 77 in the high Ct groups.Six abnormal chest CT signs were detected,namely,focal infection,patchy consolidation shadows,patchy groundglass shadows,mixed consolidation ground-glass shadows,subpleural interstitial changes,and pleural changes.Focal infections were less frequent in the low Ct group than in the medium and high Ct groups;these infections were the most common sign in the medium and high Ct groups.Patchy consolidation shadows and pleural changes were more frequent in the low Ct group than in the other two groups.The number of patients with two or more signs was greater in the low Ct group than in the medium and high Ct groups.CONCLUSION The chest CT signs of patients with pulmonary infection caused by the Omicron variants of SARSCoV-2 varied depending on the Ct values.Identification of the characteristics of Omicron variant infection can help subsequent planning of clinical treatment.
