[Objective] This study aimed to establish a TaqMan-based real-time PCR assay for detecting transmissible gastroenteritis virus （TGEV）. [Method] Primers and a probe were designed according to the conserved sequence o...[Objective] This study aimed to establish a TaqMan-based real-time PCR assay for detecting transmissible gastroenteritis virus （TGEV）. [Method] Primers and a probe were designed according to the conserved sequence of N gene in TGEV genome. After gradient dilution, the recombinant plasmid harboring the N gene was used as a standard for real-time PCR assay to establish the standard curve. [Re- sult] The results showed that the established real-time PCR assay exhibited a good linear relationship within the range of 102-10^10 copies/ul; the correlation coefficient was above 0.99 and the amplification efficiency ranged from 90% to 110%. The de- tection limit of real-time PCR assay for TGEV was 10 copies/μl, suggesting a high sensitivity; there was no cross reaction with other porcine viruses, indicating a good specificity; coefficients of variation within and among batches were lower than 3%, suggesting a good repeatability. The established real-time PCR method could be ap- plied in quantitative analysis and evaluation of the immune efficacy of TGEV vac- cines and detection of TGEV in clinical samples. [Conclusion] The TaqMan-based real-time PCR assay established in this study is highly sensitive and specific, which can provide technical means for the epidemiological survey of TGEV, development of TGEV vaccines and investigation of the pathogenesis of TGE.展开更多
This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruse...This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of ill, H3, H5, HT, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR （GM RT-PCR）. It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus （IV） rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features： high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10-5（= 280ELDs0） forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10-4（=2800 ELD50）. The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.展开更多
A polyclonal antibody-based antigen-capture ELISA （AC-ELISA） has been developed for detection of Canine parvovirus （CPV） antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as th...A polyclonal antibody-based antigen-capture ELISA （AC-ELISA） has been developed for detection of Canine parvovirus （CPV） antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1：1 600 and 1：400, respectively, in the check-board titration. In this study, a total of 152 samples （129 faecal samples and 23 cell culture supernatant） were tested both by AC-ELISA and by polymerase chain reaction （PCR）. Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 102.8 TCID50/mL whereas PCR sensitivity was 100.8 TCIDs0/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.展开更多
The etiological agent of severe acute respiratory syndrome(SARS) was identified as a new coronavirus,termed SARS-CoV. Establishment of an efficient and sensitive diagnostic system of SARS-CoV genetic materials is cruc...The etiological agent of severe acute respiratory syndrome(SARS) was identified as a new coronavirus,termed SARS-CoV. Establishment of an efficient and sensitive diagnostic system of SARS-CoV genetic materials is crucial for SARS control. In this study,we quantified SARS-CoV mRNAs in both infected cell culture lysate and in supernatant by using Real-time quantitative revere transcription-PCR based on EvaGreenTM dye and Taqman-MGB probes. For extensive evaluation of sensitivities and specificities,13 pairs of primers and 4 probes were designed based on different genes of SARS-CoV. Glyceraldehydes-3-phosphate dehydrogenase(GAPDH) was selected as the internal control gene. Results showed that S-gene-specific PCR was the most sensitive for detection,but because of its sequence variability in the different viral strains,primers and a probe based on the N gene were suitable substitutions. Meanwhile,we found the mRNA concentrations in cell culture lysates were much higher than in cell supernatant and facilited more sensitive detection of the SARS-CoV.展开更多
Arboviruses represent a serious problem to public health and agriculture worldwide. Fast, accurate identification of the viral agents of arbovirus-associated disease is essential for epidemiological surveillance and l...Arboviruses represent a serious problem to public health and agriculture worldwide. Fast, accurate identification of the viral agents of arbovirus-associated disease is essential for epidemiological surveillance and laboratory investigation. We developed a cost-effective, rapid, and highly sensitive one-step "triplex RT-PCR enzyme hybridization" assay for simultaneous detections of Japanese Encephallitis virus (JEV, Flaviviridae), Getah virus (GETV, Togaviridae), and Tahyna virus (TAHV, Bunyaviridae) using three pairs of primers to amplify three target sequences in one RT-PCR reaction. The analytical sensitivity of this assay was 1 PFU/mL for JEV, 10 PFU/mL for GETV, and 10 PFU/mL for TAHV. This assay is significantly more rapid and less expensive than the traditional serological detection and single RT-PCR reaction methods. When "triplex RT-PCR enzyme hybridization" was applied to 29 cerebrospinal fluid (CSF) samples that were JEV-positive by normal RT-PCR assay, all samples were strongly positive for JEV, but negative for GETV and TAHV, demonstrating a good sensitivity, specificity, and performance at CSF specimen detection.展开更多
A sensitive method based on solid phase PCR on oligonucleotide array was established to detect two point mutations 1896G-A and 1901G-A in hepatitis B virus (HBV) DNA, in which 6 probes including these two point mutati...A sensitive method based on solid phase PCR on oligonucleotide array was established to detect two point mutations 1896G-A and 1901G-A in hepatitis B virus (HBV) DNA, in which 6 probes including these two point mutations were immobilized on modified glass slides through 5' terminal linker, while the 3' terminal was made to be free. The mutated loci were designed to locate on the last nucleotides of 3' terminal respectively, and the positive control probes lacked the last nucleotide of 3' terminal in comparison with the probes used for detection. Probes fixed on oligonucleotide array were also the solid phase amplification primers. One pair of liquid primers was used to amplify the short template product from whole HBV DNA. Using target DNA as template, the solid primers were extended under the action of Taq DNA polymerase and incorporated with Cy-3dCTP as marker. During the thermal cycling reaction, probes served as solid phase amplification primers and amplification products bound to the oligonucleotide array, which could be visualized by incorporation with fluorescent dyes. After amplification, the oligonucleotide array was washed, performed with laser scanning, and then used for quantitative analysis to obtain the information for mutations. The hybridization results were compared with DNA sequencing. It was demonstrated that in case of sample A, the ratios of fluorescence intensities in wide type and in the mutated types of 1896G-A and 1901G-A mutations in HBV were 3.81:1 and 1:3.79 respectively, while, in case of sample B, those were 1:2.89 and 1:3.03 respectively, indicating the presence of point mutations in these two loci. These results correlated to those obtained from DNA sequencing analysis in which the fluorescence intensity ratios in wide type and in the mutated types of 1996G-A and 1901D-A mutations in HBV were 1.26:1 and 1.67:1 respectively. From the above observations, it is evident that the method using solid phase PCR based on oligonucleotide array appears to be a sensitive and promising way to detect mutations with low-density.展开更多
Noroviruses (NoVs) are widespread causes of nonbacterial gastroenteritis. Outbreaks of NoVs caused diseases are commonly ascribed to the consumption of contaminated shellfish. The concentration and RNA extraction of N...Noroviruses (NoVs) are widespread causes of nonbacterial gastroenteritis. Outbreaks of NoVs caused diseases are commonly ascribed to the consumption of contaminated shellfish. The concentration and RNA extraction of NoVs are crucial steps of detecting NoVs in shellfish. This study aimed to select a simple, rapid and highly efficient recovery method of NoVs detection with real-time RT-PCR. Four methods of recovering GI.3 and GII.4 NoVs from spiked digestive tissues of oysters and clams, respectively, were compared, of them, the method involving proteinase K and PEG 8000 was found the most efficient. With this method, 9.3% and 13.1% of GI.3 and GII.4 NoVs were recovered from oysters and 9.6% and 12.3% of GI.3 and GII.4 NoVs were recovered from clams, respectively. This method was further used to detect NoVs in 84 oysters (Crassostrea gigas) and 86 clams (Ruditapes philippinarum) collected from 10 coastal cities in China from Jan. 2011 to Feb. 2012. The NoVs isolation rates were 10.47% of clams (9/86) and 7.14% of oysters (6/84). All the detected NoVs belonged to genotype GII. The NoVs recovery method selected is efficient for NoVs detection in oysters and clams.展开更多
Ebola virus （EBOV） and Marburg virus （MARV） are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates and there is currently no licensed vaccine or therapeutics. T...Ebola virus （EBOV） and Marburg virus （MARV） are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates and there is currently no licensed vaccine or therapeutics. To date, there is no specific laboratory diagnostic test in China, while there is a national need to provide differential diagnosis during outbreaks and for instituting acceptable quarantine procedures. In this study, the TaqMan RT-PCR assays targeting the nucleoprotein genes of the Zaire Ebolavirus （ZEBOV） and MARV were developed and their sensitivities and specificities were investigated. Our results indicated that the assays were able to make reliable diagnosis over a wide range of virus copies from 103 to 109, corresponding to the threshold of a standard RNA transcript. The results showed that there were about 101 RNA copies per milliliter of virus culture supernatant, equivalent to 10,000 RNA molecules per infectious virion, suggesting the presence of many non-infectious particles. These data indicated that the TaqMan RT-PCR assays developed in this study will be suitable展开更多
OBJECTIVE Notch1 signaling has been implicated intumorigenesis. The purpose of this study was to investigate theputative role of the Notch1 receptor in carcinogenesis and in theprogression of the cervical cancer. Sinc...OBJECTIVE Notch1 signaling has been implicated intumorigenesis. The purpose of this study was to investigate theputative role of the Notch1 receptor in carcinogenesis and in theprogression of the cervical cancer. Since human papillomavirus(HPV) is a causative agent in cervical carcinoma, the interactionbetween Notch1 and HPV infection was examined.METHODS Forty cervical cancer samples and 30 normalcervical tissue specimens were examined using Western blot andRT-PCR to detect Notch1 protein and mRNA levels. HPV16 DNAwas examined in all samples using PCR.RESULTS The level of Notch1 protein expression wassignificantly lower in cervical cancer tissue than in normal tissue.Levels of Notch1 mRNA were found to be substantially downregulatedin cancer tissue. Notch1 protein expression levelswere significantly higher in carcinomas without HPV DNAthan that in carcinomas with HPV infection (55.5% vs. 3.3%, P <0.05). Down-modulation of Notch1 mRNA levels in carcinomawas demonstrated to be associated with HPVE6 transcription.Moreover, levels of Notch1 expression were shown to besignificantly higher in early stage disease than in advanced stagedisease (P = 0.001).CONCLUSION Down-modulation of Notch1 expressionprobably plays an important role in the late stages of HPVinducedcervical cancer.展开更多
The ubiquitin-like modifier bISG15 is an antiviral protein found in fetal bovine lung （FBL） cells. Bovine Herpesvirus 1（BHV-1）, which is a viral pathogen of cattle, can infect FBL cells and induce cytopathic effec...The ubiquitin-like modifier bISG15 is an antiviral protein found in fetal bovine lung （FBL） cells. Bovine Herpesvirus 1（BHV-1）, which is a viral pathogen of cattle, can infect FBL cells and induce cytopathic effects. Real-time PCR assays showed that BHV-l＇s infection could repress the basal or inducible transcription of bISG15 in FBL cells. It demonstrates that this repression effect depends on BHV-1 viral infection and new protein synthesis. Our previous work showed that bIRF-3 was the key factor in the stimulation ofbISG15 in FBL cells, so the effect of BHV-1 viral protein on bIRF-3 activating the promoter of bISG15 was confirmed. The luciferase assay showed the BHV-1 viral protein bICP0 inhibited the activation of bISG15 promoter stimulated by bIRF-3. Taken together, our work suggested that BHV-1 had some molecular mechanism to resist the cellular bISG15＇s antiviral functions.展开更多
Twenty five serotypes of Bluetongue virus (BTV) have been identified worldwide. Rapid and reliable methods of virus universal detection are essential for fighting against bluetongue (BT). We have therefore developed a...Twenty five serotypes of Bluetongue virus (BTV) have been identified worldwide. Rapid and reliable methods of virus universal detection are essential for fighting against bluetongue (BT). We have therefore developed and evaluated a pair of primers which can detect various serotypes of BTV by RT-PCR. Analysis of the viral protein 7 (VP7) and the non-structural protein (NS1) gene from different serotypes of BTV by DNAstar showed that the 5' end of the NS1 gene is the most conserved region. The primer pairs (P1 and P2) were designed based on the highly conserved region of NS1. The novel primers were evaluated by detecting BTV serotypes 1, 3, 5, 8, 10, 11, 21 and 22. The specificity of the primers was estimated by comparing to gene sequences of viruses published in GenBank, and further assessed by detecting BTV serotype 1-12 and Epizootic hemorrhagic disease virus (EHDV) serotype 1-4. The sensitivity and repeatability of PCR with the novel primers were evaluated by successfully detecting the recombinant plasmid pGEM-T121 containing the diagnosed nucleotide sequence. Our results suggest that these unique primers can be used in high throughout and universal detection of the NS1 gene from various BTV serotypes.展开更多
The objective of this study was to determine the prevalence of porcine cireovirus type 2 （PCV2） in retrospective cases of pigs associated with porcine reproductive and respiratory syndrome （PRRS）. A total of 314...The objective of this study was to determine the prevalence of porcine cireovirus type 2 （PCV2） in retrospective cases of pigs associated with porcine reproductive and respiratory syndrome （PRRS）. A total of 314 samples were collected during the period between February 2004 and August 2006 in this study, of which 196 samples were diagnosed positive for PRRS virus （PRRSV）. A total of 94 （29.9%） of 314 pigs appeared to be positive for PCV2 in the collected organ pools of lung, liver, spleen, kidney, and inguinal lymph node. However, 66 （21.0%） pigs were positive for PCV2 in the serum samples determined by PCR. The positive rate for PCV2 was much higher in PRRSV=posifive pigs than in PRRSV-negative pigs as determined by PCR from both sera and organ homogenate pools. Phylogenetic tree analysis based on the open reading frame 2 of PCV2 showed that all the PCV2 isolates in the present study shared higher nucleotide identity （98.3%-100%）, and appeared to be much closer to one of PCV2 isolates of France than others. This result indicated that PCV2 was highly prevalent in the retrospective cases of pigs in the examined areas, and the prevalence of PCV2 infection varied largely between PRRSV-positive pigs and PRRSV-negative pigs.展开更多
基金Supported by Jiangsu Agricultural Science and Technology Independent Innovation Fund[CX(13)3069]~~
文摘[Objective] This study aimed to establish a TaqMan-based real-time PCR assay for detecting transmissible gastroenteritis virus （TGEV）. [Method] Primers and a probe were designed according to the conserved sequence of N gene in TGEV genome. After gradient dilution, the recombinant plasmid harboring the N gene was used as a standard for real-time PCR assay to establish the standard curve. [Re- sult] The results showed that the established real-time PCR assay exhibited a good linear relationship within the range of 102-10^10 copies/ul; the correlation coefficient was above 0.99 and the amplification efficiency ranged from 90% to 110%. The de- tection limit of real-time PCR assay for TGEV was 10 copies/μl, suggesting a high sensitivity; there was no cross reaction with other porcine viruses, indicating a good specificity; coefficients of variation within and among batches were lower than 3%, suggesting a good repeatability. The established real-time PCR method could be ap- plied in quantitative analysis and evaluation of the immune efficacy of TGEV vac- cines and detection of TGEV in clinical samples. [Conclusion] The TaqMan-based real-time PCR assay established in this study is highly sensitive and specific, which can provide technical means for the epidemiological survey of TGEV, development of TGEV vaccines and investigation of the pathogenesis of TGE.
基金The Basic Rasearch Project of Shenzhen(JC200903190778A)
文摘This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of ill, H3, H5, HT, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR （GM RT-PCR）. It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus （IV） rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features： high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10-5（= 280ELDs0） forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10-4（=2800 ELD50）. The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.
文摘A polyclonal antibody-based antigen-capture ELISA （AC-ELISA） has been developed for detection of Canine parvovirus （CPV） antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1：1 600 and 1：400, respectively, in the check-board titration. In this study, a total of 152 samples （129 faecal samples and 23 cell culture supernatant） were tested both by AC-ELISA and by polymerase chain reaction （PCR）. Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 102.8 TCID50/mL whereas PCR sensitivity was 100.8 TCIDs0/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.
基金The Knowledge Innovation Program of Chinese Academy of Sciences (KSCX2-YW-N-065)FP6 projects DISSECT (N°SP22-CT-2004-511060)EPIS-ARS (N°SP22-CT-2004-511603)
文摘The etiological agent of severe acute respiratory syndrome(SARS) was identified as a new coronavirus,termed SARS-CoV. Establishment of an efficient and sensitive diagnostic system of SARS-CoV genetic materials is crucial for SARS control. In this study,we quantified SARS-CoV mRNAs in both infected cell culture lysate and in supernatant by using Real-time quantitative revere transcription-PCR based on EvaGreenTM dye and Taqman-MGB probes. For extensive evaluation of sensitivities and specificities,13 pairs of primers and 4 probes were designed based on different genes of SARS-CoV. Glyceraldehydes-3-phosphate dehydrogenase(GAPDH) was selected as the internal control gene. Results showed that S-gene-specific PCR was the most sensitive for detection,but because of its sequence variability in the different viral strains,primers and a probe based on the N gene were suitable substitutions. Meanwhile,we found the mRNA concentrations in cell culture lysates were much higher than in cell supernatant and facilited more sensitive detection of the SARS-CoV.
基金NIH Grant (2U54AI057160-06)Development Grant of State Key Laboratory for Infectious Disease Prevention and Control (2008SKLID105)
文摘Arboviruses represent a serious problem to public health and agriculture worldwide. Fast, accurate identification of the viral agents of arbovirus-associated disease is essential for epidemiological surveillance and laboratory investigation. We developed a cost-effective, rapid, and highly sensitive one-step "triplex RT-PCR enzyme hybridization" assay for simultaneous detections of Japanese Encephallitis virus (JEV, Flaviviridae), Getah virus (GETV, Togaviridae), and Tahyna virus (TAHV, Bunyaviridae) using three pairs of primers to amplify three target sequences in one RT-PCR reaction. The analytical sensitivity of this assay was 1 PFU/mL for JEV, 10 PFU/mL for GETV, and 10 PFU/mL for TAHV. This assay is significantly more rapid and less expensive than the traditional serological detection and single RT-PCR reaction methods. When "triplex RT-PCR enzyme hybridization" was applied to 29 cerebrospinal fluid (CSF) samples that were JEV-positive by normal RT-PCR assay, all samples were strongly positive for JEV, but negative for GETV and TAHV, demonstrating a good sensitivity, specificity, and performance at CSF specimen detection.
文摘A sensitive method based on solid phase PCR on oligonucleotide array was established to detect two point mutations 1896G-A and 1901G-A in hepatitis B virus (HBV) DNA, in which 6 probes including these two point mutations were immobilized on modified glass slides through 5' terminal linker, while the 3' terminal was made to be free. The mutated loci were designed to locate on the last nucleotides of 3' terminal respectively, and the positive control probes lacked the last nucleotide of 3' terminal in comparison with the probes used for detection. Probes fixed on oligonucleotide array were also the solid phase amplification primers. One pair of liquid primers was used to amplify the short template product from whole HBV DNA. Using target DNA as template, the solid primers were extended under the action of Taq DNA polymerase and incorporated with Cy-3dCTP as marker. During the thermal cycling reaction, probes served as solid phase amplification primers and amplification products bound to the oligonucleotide array, which could be visualized by incorporation with fluorescent dyes. After amplification, the oligonucleotide array was washed, performed with laser scanning, and then used for quantitative analysis to obtain the information for mutations. The hybridization results were compared with DNA sequencing. It was demonstrated that in case of sample A, the ratios of fluorescence intensities in wide type and in the mutated types of 1896G-A and 1901G-A mutations in HBV were 3.81:1 and 1:3.79 respectively, while, in case of sample B, those were 1:2.89 and 1:3.03 respectively, indicating the presence of point mutations in these two loci. These results correlated to those obtained from DNA sequencing analysis in which the fluorescence intensity ratios in wide type and in the mutated types of 1996G-A and 1901D-A mutations in HBV were 1.26:1 and 1.67:1 respectively. From the above observations, it is evident that the method using solid phase PCR based on oligonucleotide array appears to be a sensitive and promising way to detect mutations with low-density.
基金supported by the China-Australia Bilateral Research Program (No. 2010DFA31720)the National Key Technology R&D Program (2012BAD28B05)
文摘Noroviruses (NoVs) are widespread causes of nonbacterial gastroenteritis. Outbreaks of NoVs caused diseases are commonly ascribed to the consumption of contaminated shellfish. The concentration and RNA extraction of NoVs are crucial steps of detecting NoVs in shellfish. This study aimed to select a simple, rapid and highly efficient recovery method of NoVs detection with real-time RT-PCR. Four methods of recovering GI.3 and GII.4 NoVs from spiked digestive tissues of oysters and clams, respectively, were compared, of them, the method involving proteinase K and PEG 8000 was found the most efficient. With this method, 9.3% and 13.1% of GI.3 and GII.4 NoVs were recovered from oysters and 9.6% and 12.3% of GI.3 and GII.4 NoVs were recovered from clams, respectively. This method was further used to detect NoVs in 84 oysters (Crassostrea gigas) and 86 clams (Ruditapes philippinarum) collected from 10 coastal cities in China from Jan. 2011 to Feb. 2012. The NoVs isolation rates were 10.47% of clams (9/86) and 7.14% of oysters (6/84). All the detected NoVs belonged to genotype GII. The NoVs recovery method selected is efficient for NoVs detection in oysters and clams.
基金Supported by Important National Science&Technology Specific Projects(2009ZX10004-504,2009ZX09301-014)National Natural Science Foundation of China(81072675)
文摘Ebola virus （EBOV） and Marburg virus （MARV） are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates and there is currently no licensed vaccine or therapeutics. To date, there is no specific laboratory diagnostic test in China, while there is a national need to provide differential diagnosis during outbreaks and for instituting acceptable quarantine procedures. In this study, the TaqMan RT-PCR assays targeting the nucleoprotein genes of the Zaire Ebolavirus （ZEBOV） and MARV were developed and their sensitivities and specificities were investigated. Our results indicated that the assays were able to make reliable diagnosis over a wide range of virus copies from 103 to 109, corresponding to the threshold of a standard RNA transcript. The results showed that there were about 101 RNA copies per milliliter of virus culture supernatant, equivalent to 10,000 RNA molecules per infectious virion, suggesting the presence of many non-infectious particles. These data indicated that the TaqMan RT-PCR assays developed in this study will be suitable
文摘OBJECTIVE Notch1 signaling has been implicated intumorigenesis. The purpose of this study was to investigate theputative role of the Notch1 receptor in carcinogenesis and in theprogression of the cervical cancer. Since human papillomavirus(HPV) is a causative agent in cervical carcinoma, the interactionbetween Notch1 and HPV infection was examined.METHODS Forty cervical cancer samples and 30 normalcervical tissue specimens were examined using Western blot andRT-PCR to detect Notch1 protein and mRNA levels. HPV16 DNAwas examined in all samples using PCR.RESULTS The level of Notch1 protein expression wassignificantly lower in cervical cancer tissue than in normal tissue.Levels of Notch1 mRNA were found to be substantially downregulatedin cancer tissue. Notch1 protein expression levelswere significantly higher in carcinomas without HPV DNAthan that in carcinomas with HPV infection (55.5% vs. 3.3%, P <0.05). Down-modulation of Notch1 mRNA levels in carcinomawas demonstrated to be associated with HPVE6 transcription.Moreover, levels of Notch1 expression were shown to besignificantly higher in early stage disease than in advanced stagedisease (P = 0.001).CONCLUSION Down-modulation of Notch1 expressionprobably plays an important role in the late stages of HPVinducedcervical cancer.
基金National Natural Science Foundation of China (81071343) and (30970162)
文摘The ubiquitin-like modifier bISG15 is an antiviral protein found in fetal bovine lung （FBL） cells. Bovine Herpesvirus 1（BHV-1）, which is a viral pathogen of cattle, can infect FBL cells and induce cytopathic effects. Real-time PCR assays showed that BHV-l＇s infection could repress the basal or inducible transcription of bISG15 in FBL cells. It demonstrates that this repression effect depends on BHV-1 viral infection and new protein synthesis. Our previous work showed that bIRF-3 was the key factor in the stimulation ofbISG15 in FBL cells, so the effect of BHV-1 viral protein on bIRF-3 activating the promoter of bISG15 was confirmed. The luciferase assay showed the BHV-1 viral protein bICP0 inhibited the activation of bISG15 promoter stimulated by bIRF-3. Taken together, our work suggested that BHV-1 had some molecular mechanism to resist the cellular bISG15＇s antiviral functions.
基金Hi-Tech Research and Development Program of China (2006AA10Z446)
文摘Twenty five serotypes of Bluetongue virus (BTV) have been identified worldwide. Rapid and reliable methods of virus universal detection are essential for fighting against bluetongue (BT). We have therefore developed and evaluated a pair of primers which can detect various serotypes of BTV by RT-PCR. Analysis of the viral protein 7 (VP7) and the non-structural protein (NS1) gene from different serotypes of BTV by DNAstar showed that the 5' end of the NS1 gene is the most conserved region. The primer pairs (P1 and P2) were designed based on the highly conserved region of NS1. The novel primers were evaluated by detecting BTV serotypes 1, 3, 5, 8, 10, 11, 21 and 22. The specificity of the primers was estimated by comparing to gene sequences of viruses published in GenBank, and further assessed by detecting BTV serotype 1-12 and Epizootic hemorrhagic disease virus (EHDV) serotype 1-4. The sensitivity and repeatability of PCR with the novel primers were evaluated by successfully detecting the recombinant plasmid pGEM-T121 containing the diagnosed nucleotide sequence. Our results suggest that these unique primers can be used in high throughout and universal detection of the NS1 gene from various BTV serotypes.
文摘The objective of this study was to determine the prevalence of porcine cireovirus type 2 （PCV2） in retrospective cases of pigs associated with porcine reproductive and respiratory syndrome （PRRS）. A total of 314 samples were collected during the period between February 2004 and August 2006 in this study, of which 196 samples were diagnosed positive for PRRS virus （PRRSV）. A total of 94 （29.9%） of 314 pigs appeared to be positive for PCV2 in the collected organ pools of lung, liver, spleen, kidney, and inguinal lymph node. However, 66 （21.0%） pigs were positive for PCV2 in the serum samples determined by PCR. The positive rate for PCV2 was much higher in PRRSV=posifive pigs than in PRRSV-negative pigs as determined by PCR from both sera and organ homogenate pools. Phylogenetic tree analysis based on the open reading frame 2 of PCV2 showed that all the PCV2 isolates in the present study shared higher nucleotide identity （98.3%-100%）, and appeared to be much closer to one of PCV2 isolates of France than others. This result indicated that PCV2 was highly prevalent in the retrospective cases of pigs in the examined areas, and the prevalence of PCV2 infection varied largely between PRRSV-positive pigs and PRRSV-negative pigs.
