Establishment of a TaqMan-MGB probe multiplex real-time PCR system for one-step levofloxacin and clarithromycin resistant Helicobacter pylori detection.

Due to the abuse of antibiotics, the prevalence of antibiotic resistant Helicobacter pylori strains continues to increase. Therefore, antibiotic resistance assessment is now essential in addition to general H. pylori diagnosis in medical institutions to fulfill clinicians administering effective antibiotic regimens. However, the conventional antibiotic resistance assessment methods, such as in vitro antibiotic susceptibility test and E-test, are skilled-staff dependent and time-consuming. The aim of this study was to establish an easy-operating TaqMan-MGB probe multiplex real-time PCR system for one-step detection of levofloxacin and clarithromycin resistance mutations with concurrent H. pylori infection diagnosis. Through the optimization of primers, probes and reaction buffers, this proposed system could accurately distinguish the recombinant plasmids with different mutation markers. More importantly, the diagnosis results of this detection system exhibited excellent consistence with the gold standard of gastric biopsy and Sanger sequencing on the detection of H. pylori infection and relevant antibiotic resistant strains, the Kappa values of which all exceeded 0.90. In addition, the results of this detection system could also be applied for the prevalence statistics of antibiotic resistance patterns for patients by age, gender and geographical location. This simple and rapid system should be beneficial for clinicians issuing personalized treatments according to the patient's H. pylori strains and avoid the abuse of antibiotics.

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.

A TaqMan-MGB real-time RT-PCR assay with an internal amplification control for rapid detection of Muscovy duck reovirus.

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of Muscovy duck reovirus (MDRV) RNA in clinical samples is described. The assay is based on TaqMan-MGB technology, consisting of two primers and one probe labeled with the reporter dye 6-carboxyfluorescein that binds selectively to the sigma B-protein gene of MDRV. This technique also includes an Internal Positive Control (IPC). The real-time RT-PCR assay was able to detect MDRVs, whereas other common waterfowl-origin viral pathogens were not recognised by the established oligonucleotide set, thus showing that the test was specific for MDRV. The sensitivity of the assay was 2.83 × 101 copies/µL and was 100 times higher than that of the conventional RT-PCR. The variation coefficients of intra-assay and inter-assay were less than 1.5% which verified sufficient repeatability of this assay. The use of ß-actin mRNA as an IPC in order not to reduce the efficiency of the assay was adopted. The detection for 100 clinical samples showed that the positive rate of the established TaqMan-MGB real-time RT-PCR method was 87% (87/100), while the positive rate of the conventional RT-PCR was 83% (83/100), with the coincidence rate was 97.14%. Sensitivity and positive rate for clinical samples of TaqMan fluorescent quantitative RT-PCR were higher than conventional RT-PCR. The high specificity, sensitivity, and rapidity TaqMan-MGB real-time RT-PCR assay with the use of IPC to monitor for false negative results can make this method suitable for the pathogenic surveillance and epidemiological investigation of MDRV infection.

Duplex real-time PCR assay for the simultaneous detection of Caliciopsis pinea and Fusarium circinatum in pine samples.

Fusarium circinatum and Caliciopsis pinea are the causal agents of Pitch canker and Caliciopsis canker, respectively. These diseases affect pines and other conifers both in Europe and North America. The two pathogens cause similar bleeding cankers, especially at the early stage of colonization. Symptoms closely resembling those due to F. circinatum can be instead associated with C. pinea. Since F. circinatum is a quarantine organism, subjected to provisional emergency measures, its report immediately causes serious economic implications, while C. pinea, even if now emerging, is not regulated in the EU nor in the USA. For this reason, a reliable and accurate diagnostic tool able to distinguish between the two organisms was considered a priority. In this study, we developed and standardized a duplex real-time PCR assay allowing the simultaneous recognition of C. pinea and F. circinatum DNA in pine tissue in a reasonably short time and for amounts as small as 0.06 pg/µl. The molecular assay is, therefore, able to detect the infection even before symptoms have fully developed. The test was challenged with a very large set of strains (110 different isolates) collected in different regions of the world and host trees, and gave reliable results. The high efficiency of this method suggests its use as a standard diagnostic tool during phytosanitary controls. In addition, the duplex real-time PCR assay presented here is the first DNA-based method designed to detect C. pinea, which is becoming an increasing threat to pine stands both in North America and in Europe.

Detection of KIT Genotype in Pigs by TaqMan MGB Real-Time Quantitative Polymerase Chain Reaction.

The dominant white phenotype in domestic pigs is caused by two mutations in the KIT gene: a 450 kb duplication containing the entire KIT gene together with flanking sequences and one splice mutation with a G:A substitution in intron 17. The purpose of this study was to establish a simple, rapid method to determine KIT genotype in pigs. First, to detect KIT copy number variation (CNV), primers for exon 2 of the KIT gene, along with a TaqMan minor groove binder (MGB) probe, were designed. The single-copy gene, estrogen receptor (ESR), was used as an internal control. A real-time fluorescence-based quantitative PCR (FQ-PCR) protocol was developed to accurately detect KIT CNVs. Second, to detect the splice mutation ratio of the G:A substitution in intron 17, a 175 bp region, including the target mutation, was amplified from genomic DNA. Based on the sequence of the resulting amplified fragment, an MGB probe set was designed to detect the ratio of splice mutation to normal using FQ-PCR. A series of parallel amplification curves with the same internal distances were obtained using gradually diluted DNA as templates. The CT values among dilutions were significantly different (p < 0.001) and the coefficients of variation from each dilution were low (from 0.13% to 0.26%). The amplification efficiencies for KIT and ESR were approximately equal, indicating ESR was an appropriate control gene. Furthermore, use of the MGB probe set resulted in detection of the target mutation at a high resolution and stability; standard curves illustrated that the amplification efficiencies of KIT1 (G) and KIT2 (A) were approximately equal (98.8% and 97.2%). In conclusion, a simple, rapid method, with high specificity and stability, for the detection of the KIT genotype in pigs was established using TaqMan MGB probe real-time quantitative PCR.

Development of an RT-qPCR assay for the specific detection of a distinct genetic lineage of the infectious bursal disease virus.

The infectious bursal disease virus (IBDV) is a major health threat to the world's poultry industry despite intensive controls including proper biosafety practices and vaccination. IBDV (Avibirnavirus, Birnaviridae) is a non-enveloped virus with a bisegmented double-stranded RNA genome. The virus is traditionally classified into classic, variant and very virulent strains, each with different epidemiological relevance and clinical implications. Recently, a novel worldwide spread genetic lineage was described and denoted as distinct (d) IBDV. Here, we report the development and validation of a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay for the specific detection of dIBDVs in the global poultry industry. The assay employs a TaqMan-MGB probe that hybridizes with a unique molecular signature of dIBDV. The assay successfully detected all the assessed strains belonging to the dIBDV genetic lineage, showing high specificity and absence of cross-reactivity with non-dIBDVs, IBDV-negative samples and other common avian viruses. Using serial dilutions of in vitro-transcribed RNA we obtained acceptable PCR efficiencies and determination coefficients, and relatively small intra- and inter-assay variability. The assay demonstrated a wide dynamic range between 103 and 108 RNA copies/reaction. This rapid, specific and quantitative assay is expected to improve IBDV surveillance and control worldwide and to increase our understanding of the molecular epidemiology of this economically detrimental poultry pathogen.

A misleading false-negative result of Pneumocystis real-time PCR assay due to a rare punctual mutation: A French multicenter study.

This article describes a previously unreported mutation at position 210 (C210T) of the mitochondrial large subunit ribosomal RNA (mtLSUrRNA) gene of Pneumocystis jirovecii, which led to a false-negative result of a real-time polymerase chain reaction (PCR) assay. Since the aforementioned real-time PCR assay is widely used in France, a French multicenter study was conducted to estimate the mutation frequency and its potential impact on the routine diagnosis of Pneumocystis pneumonia (PCP). Through analysis of data obtained from eight centers, the mutation frequency was estimated at 0.28%. This low frequency should not call into question the routine use of this PCR assay. Nonetheless, the occurrence of the false-negative PCR result provides arguments for maintaining microscopic techniques combined to PCR assays to achieve PCP diagnosis.

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.

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.

CYP2E1 gene rs6413420 polymorphism was first found in the Bouyei ethnic group of China.

BACKGROUND: China is a multinational country. The relationship between gene polymorphisms of xenobiotic metabolizing enzymes and national ethnicity has not previously investigated among Chinese people. The aim of this study was to investigate distributions of CYP1A1 and CYP2E1 gene polymorphisms in five ethnic groups of China. METHODS: 829 blood samples were collected from five ethnic groups (Han, Shui, Miao, Zhuang, Bouyei). Taqman-MGB probe was used in Real-time PCR to test the gene polymorphisms of CYP1A1 (rs1048943 and rs4646903) and CYP2E1 (rs2031920 and rs6413420). We further validate the SNP genotyping results through DNA sequencing. RESULTS: The genotype distribution of all four SNPs was in accordance with Hardy-Weinberg equilibrium except the genotype distribution of rs4646903 in Han and Bouyei ethnic groups (p=0.013 and 0.0005, respectively). CYP2E1 gene rs6413420 polymorphism was first found in the Bouyei ethnic group in China. The results of DNA sequencing were entirely in line with the SNP genotyping assay. CONCLUSIONS: The CYP1A1 and CYP2E1 genetic polymorphisms were different in different ethnic groups in China. CYP2E1 gene rs6413420 polymorphism was first found in the Bouyei ethnic group of China.

Detection and quantification by PCR assay of the biocontrol agent Pantoea agglomerans CPA-2 on apples.

The registration of biological control agents requires the development of monitoring systems to detect and quantify the agent in the environment. Pantoea agglomerans CPA-2 is an effective biocontrol agent for postharvest diseases of citrus and pome fruits. The monitoring of CPA-2 in postharvest semi-commercial trials was evaluated by Rodac impression plates and the colonies isolated were confirmed by conventional PCR using the SCAR primers PAGA1 and PAGB1. Samples were taken from different surfaces that had contact with CPA-2, the surrounding environment and working clothes worn by handlers. Moreover, population dynamics of the strain CPA-2 were determined on apple surfaces using both the classical plating technique and real-time quantitative PCR (qPCR). A qPCR assay using a 3'-minor groove-binding (MGB) probe was developed for the specific detection and quantification of P. agglomerans strain CPA-2. Based on the nucleotide sequence of a SCAR fragment of CPA-2, one primer set and TaqMan MGB probe were designed. The primers SP2-F/SP2-R and the TaqMan MGB probe showed a specific detection of strain CPA-2 on apple surfaces, which was verified tested against purified DNA from 17 strains of P. agglomerans, 4 related Pantoea species, and 21 bacterial strains from other genera isolated from whole and also freshly-cut fruit and vegetables. The detection level was approximately 10(3) cells per reaction, and the standard curve was linear within a range of 5log units. Results from semi-commercial trials showed that CPA-2 had a low impact. The maximum persistence of P. agglomerans CPA-2 was not longer than 5days in plastic boxes stored at 0°C. Significant differences in CPA-2 population level dynamics were observed in results obtained by qPCR and dilution plating. These differences may indicate the presence of non-degraded DNA from non-viable cells. In conclusion, qPCR is a novel potential tool to quickly and specifically monitor recent surface colonisation by CPA-2 populations on apple surfaces during large-scale experiments that could ensure efficient and successful treatments.

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.

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.