Identification of Fritillaria taibaiensis and its relatives by real-time PCR with a TaqMan-MGB probe / 药学学报

The molecular identification of Fritillaria taibaiensis and its relatives was studied by real-time PCR with a TaqMan-MGB probe. DNA was extracted from F. taibaiensis and its relatives. According to the sequence of ITS1 region, the mutation sites of F. taipaiensis and its related species were identified by MEGA7.0 software. The specific primers (a pair) and a TaqMan-MGB probe were designed by Primer Premier 6.0 software. In the Roche LightCycler 96 system, the lowest limit of detection for F. taipaiensis DNA template was 0.002 39 ng·μL-1, and the optimal Tm value range was 60 and 61 ℃. Specificity identification showed that the method had good specificity for F. taipaiensis, as it could be distinguished from other 13 different Fritillaria species including F. unibracteata. Since this method could accurately identify F. taipaiensis and its related species, it provides technical support for rational development of F. taipaiensis resources, management of Chinese medicinal market and supervision of raw materials in Chinese medicine manufacturing enterprises.

Development of Fluorescence PCR for Detection of Hand Foot Mouth Viruses with MGB Probe / 现代检验医学杂志

Objective A fluorescence PCR methods was developed to detect EV71 and CoxAl6 and other enteroviruses simultaneously,which used for hand,foot and mouth disease (HFMD) viruses in the clinical rapid diagnosis.Methods Designed specific primers and probes of the enteroviruses gene which represents one of the highly conserved regions of the virus gene,and optimized the detection system of real-time quantitative RT-PCR.The positive control template and the standard curve were constructed.Researched the limit of detection,repeatability and specificity of the products,and tested 26 positive samples and 10 negative samples which collected on June 2015.Results The results showed that this experiment obtained positive recombinant plasmid,the range of the linear relation was from 8 × 102 to 8 × 108 copies/μl,and the detection result within this range was fine.The optimal concentrations of EVUN upstream and downstream primers were 0.50 μmol/L and the MGB probes were 0.30 μmol/L,RT-PCR reaction conditions as follows:42℃ 30 min,95℃ 3 min.95℃ 5 s,60℃ 35 s,45 cycle.The limit of detection reached to 800 copies/μl.The CV of the repeatability assay was no more than 5％.Specificity was good,and no cross reaction with other infectious viruses.26 clinical positive samples and 10 negative samples were detected in this experiment,detection rate of positive samples was 100 ％ (26/26) and negative samples was 100 ％ (10/10) by fluorescent quantitative RT-PCR detection method.Conclusion The experiment demonstrated that the detection method of the fluorescent quantitative PCR for hand-foot-mouth viruses could be used for the rapid diagnosis in clinical application.

Development and application of dual real time RT-PCR for avian influenza H5N6 virus / 中华实验和临床病毒学杂志

Objective@#To establish a TaqMan-MGB probe-based real-time fluorescence RT-PCR assay for avian influenza H5N6 virus used in rapid diagnosis for suspected cases and surveillance for outer environment of live poultry markets.@*Methods@#Based on the conservative sequences of avian influenza H5N6 virus for HA and NA gene published on GenBank, specific primers and TaqMan-MGB probes were designed to develop and optimize for the dual real-time RT-PCR assay. Specificity, sensitivity, repeatability and comparison tests were carried out.@*Results@#This dual real-time RT-PCR detection can be completed within 80 minutes. There was no cross-reaction with other subtypes of influenza virus and common respiratory pathogens. The minimum detection limit could be up to 10 copies/reaction. The correlation coefficient of standard curve for the gene of H5 and N6 were 0.999 and 0.993, and the coefficients of variation for cycle threshold were range from 0.151%-0.549%and 0.213%-0.575%, respectively. The positive and negative coincidence rates of the validation test were 100%.@*Conclusions@#This TaqMan-MGB probe-based dual real-time RT-PCR for avian influenza H5N6 virus was rapid, specific and sensitive. It will have a good use in early emergency detection of suspected cases and continuous monitoring of external environment in live poultry trade market.

Development of a New TaqMan-MGB Probe-Based Real-Time qPCR Method for the Detection of HCV-RNA / 现代检验医学杂志

Objective Developing a rapid and accurate real-time qPCR method for the detection of HCV-RNA.Methods HCV nucleotide sequence was analysed in Clustal software and primers and probe were designed in the conserved region of 5'UTR.The reaction system optimization of real-time qPCR method was used chessboard titration,pseudoviral particles were used as quantitative standard to assess the performance.New methods was compared with clinical commonly used kit of HCV-RNA and discuss the application value.Results The sensitivity of new real-time qPCR method was 50 IU/ml,coefficient variation was less than 5％.The quantitative results of this method could be traceable to national standards of GBW09151a.40 samples were determined by new methods and clinical commonly used kit of HCV-RNA,the positive concordance rate was 100 ％,the negative concordance rate was 56 ％.14 samples were positive by new method,but negative by Qiagen kit,illustrating that the sensitivity of new method was superior to Qiagen kit.Conclusion New TaqMan-MGB probe-based real-time qPCR method is a specific,sensitive,simple,rapid and exactly used to detection of HCV-RNA.

Using the real-time PCR assay to establish TaqMan-MGB probe for rapid identification of Clostridium difficile and its toxin / 中华流行病学杂志

Objective To develop a real-time PCR assay for the rapid identification of Clostridium(C.)difficile and its toxin. Methods TaqMan real-time PCR was developed for the rapid identification of species specific gene(tpi)of C. difficile strains and the toxins A(TcdA),B(TcdB) and truncated toxin A(TcdAT). Sensitivity,specificity and anti-interference ability of these methods were estimated,as well. Feces sampled from fifty diarrhea patients were tested by real-time PCR and compared to the results from VIDAS assay. Results The detection limits of tpi were 6×10-2 CFU/μl and 6 × 10-1 CFU/μl in the non-oxin producing and toxin producing strains,respectively. The coefficients of variability (CV) of intra-assay and inter-assay for the detection limits of tpi in the non-toxin producing strain were 2.1% and 2.3%. The CVs of intra-assay and inter-assay for the detection limit of tpi,tcdA,tcdB and tcdAT in the toxin producing strain were 3.0%and 3.4%,2.9%and 3.2%,5.3%and 5.7%,2.7%and 2.8%,respectively. No interferance was detected from other genus or species in clostridium. From 50 clinical samples,thirty-nine of them were negative and six of them were positive under the TaqMan-MGB probe technique in accordance with VIDAS. Five samples appeared positive using the TaqMan-MGB probe technique,in which 3 were dubious and 2 were negative under VIDAS. Conclusion The newly developed method was a sensitive and reliable assay for rapid identification of C. difficile and its toxin. This method could be used to screen C. difficile isolates harboring truncated toxin A to avoid misdiagnosis,clinically.

Using the real-time PCR assay to establish TaqMan-MGB probe for rapid identification of Clostridium difficile and its toxin / 中华流行病学杂志

Objective To develop a real-time PCR assay for the rapid identification of Clostridium(C.)difficile and its toxin. Methods TaqMan real-time PCR was developed for the rapid identification of species specific gene(tpi)of C. difficile strains and the toxins A(TcdA),B(TcdB) and truncated toxin A(TcdAT). Sensitivity,specificity and anti-interference ability of these methods were estimated,as well. Feces sampled from fifty diarrhea patients were tested by real-time PCR and compared to the results from VIDAS assay. Results The detection limits of tpi were 6×10-2 CFU/μl and 6 × 10-1 CFU/μl in the non-oxin producing and toxin producing strains,respectively. The coefficients of variability (CV) of intra-assay and inter-assay for the detection limits of tpi in the non-toxin producing strain were 2.1% and 2.3%. The CVs of intra-assay and inter-assay for the detection limit of tpi,tcdA,tcdB and tcdAT in the toxin producing strain were 3.0%and 3.4%,2.9%and 3.2%,5.3%and 5.7%,2.7%and 2.8%,respectively. No interferance was detected from other genus or species in clostridium. From 50 clinical samples,thirty-nine of them were negative and six of them were positive under the TaqMan-MGB probe technique in accordance with VIDAS. Five samples appeared positive using the TaqMan-MGB probe technique,in which 3 were dubious and 2 were negative under VIDAS. Conclusion The newly developed method was a sensitive and reliable assay for rapid identification of C. difficile and its toxin. This method could be used to screen C. difficile isolates harboring truncated toxin A to avoid misdiagnosis,clinically.

Develonment and application of TaqMan MGB probe real-time fluorescence quantitative PCR for rapid detection of Helicobacter hepaticus / 中华微生物学和免疫学杂志

Objective To develop a TaqMan MGB probe-based,sensitive and specific real-time fluorescence quantitative PCR assay for rapid detection of Helicobacter hepaticus.Methods Primers and probes specific toflaB gene of Helicobacter hepaticus were designed.A TaqMan MGB probe-based,real-time fluorescence quantitative PCR was established.The specificity,sensitivity and stability of the assay were assassed.Then,the established TaqMan MGB probe real-time fluorescence quantitative PCR assay was applied to detect Helicobacter hepaticus in 1081 clinical specimens during 2008-2011,compared with bacterial isolation and culture method and conventional PCR assay.Results The specificity of this established TaqMan MGB probe-based real-time fluorescence quantitative PCR was high and there were no cross-reactivity with Helicobacter pylori,Campylobacter jejuni,Clostridium piliforme,Pasteurella pneumotropica,Escherichia coli,Pseudomonas aeruginosa.The detection limits was 8.3 copies.The correlation coefficient and slope value of standard curve were 0.999 and -3.227,respectively and the efficiency of TaqMan MGB-based probe realtime fluorescence quantitative PCR assay was 100％.The TaqMan MGB-based probe real-time fluorescence quantitative PCR and conventional PCR were preformed to detect Helicobacter hepaticus in 1081 clinical specimens,a total of 86 specimens were positive for Helicobacter hepaticus.However,there was only 4 specimens were positive by bacteria isolation and culture method.The results showed that TaqMan MGB -based probe real-time fluorescence quantitative PCR for Helicobacter hepaticas was more sensitive than bacteria isolation and culture method,and it could detect Helicobacter hepaticus DNA from clinical specimens directly,and detection time is only 2 hours.Conclusion The TaqMan MGB-based probe real-time fluorescence quantitative PCR assay was a reliable,specific,sensitive and useful tool for rapid detection of Helicobacter hepaticus.

Research on detection of Legionella pneumophila by real-time fluorescence PCR / 中华微生物学和免疫学杂志

Objective To develop a real-time fluorescence quantitative PCR method for detection of Legionella pneumophila as a tool for environmental and clinical examination. Methods A pair of degen-erated primers and one TaqMan-MGB probe were designed to test the conserved region at the macrophage in-fective potentiation (mip) gene of Legionella pneumophila. TaqMan MGB real-time quantitative PCR assay was developed with pMD-19T plasmid including mip gene of Legionella pneumophila as standard sample. The sensitivity and specificity of the real-time quantitative PCR was evaluated using the standard sample and dif-ferent strains. Results The detection limit of 0.71 copy/μl was obtained for the standard sample in a reac-tion system of 0.6μl of sense and antisense primers (20μmol/L), respectively, 0.4μl of probe (20μmol/L) and 6.0μl of DNA temple. Conditions for the PCR reactions were as follows. After an initial de-naturation at 95℃ for 20s, 40 amplification cycles were performed. Each cycle consisted of denaturation at 95℃ for 10 s, primer annealing at 50℃ for45s. The PCR Ct value of a standard strain and 12 isolates was in a scale of 13.23 and 16.04. However, the Ct values of the strains of Staphylococcus aureus, Salmonella typhimurium, Vibrio parahaemolyticus, Escherichia coli, Pseudomonas aeruginosa and Shigella sonnei were greater than 30. Conclusion The real-time quantitative PCR method has good sensitivity and specificity and the result has potentiality of applying for detecting Legionella pneumophila.

Detection of Double Mutation of Basal Core Promoter(BCP) of HBV by TaqMan MGB / 中华医院感染学杂志

OBJECTIVE To establish a simple,sensitive method for detecting the double mutation of the basal core promoter(BCP) of HBV.METHODS FAM fluorescence-labeled TaqMan MGB and primers driving from the region containing the double mutation of BCP were designed for the real time PCR,then the standard positive control,standard negative control and HBV DNA were amplified and detected by the real time PCR.The results of detecting the double mutation of BCP were validated by the direct-sequencing analysis of PCR products.RESULTS The double mutation of BCP of HBV could be detected by the real time PCR.The sensitivity of the method was 3?100 copy templates and as few as 1% of mutant among wild-type virus sequence were detected.CONCLUSIONS The method can be used to detect the double mutation of BCP of serum HBV DNA.