transcription-PCR Assay For Detection Research Articles

Development of one-step reverse transcription PCR assay for detection of porcine epidemic diarrhoea virus in pigs

Development of one-step reverse transcription PCR assay for detection of porcine epidemic diarrhoea virus in pigs

Development and clinical validation of a one-step pentaplex real-time reverse transcription PCR assay for detection of hepatitis virus B, C, E, Treponema pallidum, and a human housekeeping gene

BackgroundWith the safety of blood transfusion being a major public health concern, the development of a rapid, sensitive, specific, and cost-effective multiplex PCR assay for simultaneous detection of hepatitis B virus(HBV), hepatitis C virus (HCV), hepatitis E virus (HEV), and Treponema pallidum(T. pallidum) in blood is crucial.MethodsFive primer pairs and probes were designed towards conserved regions of target genes and used to establish a one-step pentaplex real-time reverse transcription PCR(qRT-PCR) assay for simultaneous detection of HBV, HCV, HEV, T. pallidum, and RNase P(housekeeping gene), providing sample quality check. The clinical performance of the assay was further determined with 2400 blood samples from blood donors and patients in Zhejiang province, and compared the results with commercial singleplex qPCR and serological assays.ResultsThe 95% limit of detection(LOD) of HBV, HCV, HEV, and T. pallidum were 7.11 copies/µL, 7.65 copies/µL, 8.45 copies/µL, and 9.06 copies/µL, respectively. Moreover, the assay has good specificity and precision. Compared to the singleplex qPCR assay, the novel assay for detecting HBV, HCV, HEV, and T. pallidum presented 100% clinical sensitivity, specificity, and consistency. Several discrepant results between serological and pentaplex qRT-PCR assays were found. Of 2400 blood samples, there were 2(0.08%) HBsAg positive samples, 3(0.13%) anti-HCV positive samples, 29(1.21%) IgM anti-HEV positive samples and 6(0.25%) anti-T. pallidum positive samples proven negative in nucleic acid detection. 1(0.04%) HBV DNA positive sample and 1(0.04%) HEV RNA positive sample were detected negative by serological testing.ConclusionsThe developed pentaplex qRT-PCR is the first assay on simultaneous, sensitive, specific, and reproducible detection of HBV, HCV, HEV, T. pallidum, and RNase P in a single tube. It could detect pathogens in blood during the window period of infection and is a good tool for effectively screening blood donors and early clinical diagnosis.

Development and application of SYBR Green Ⅰ real-time quantitative reverse transcription PCR assay for detection of swine Getah virus

Getah virus (GETV), a mosquito-borne virus belonging to the Alphavirus genus of family Togaviridae, has become increasingly problematic, which poses a huge threat to the safety of animals and public health. In order to detect GETV quickly and accurately, we have developed a SYBR Green I real-time quantitative reverse transcription PCR (RT-qPCR) assay for GETV with the detection limit of 66 copies/μL, excellent correlation coefficient (R2) of 0.9975, and amplification efficiency (E) of 98.90%, the target selected was the non-structural protein 3 of GETV. The sensitivity of it was higher than that of ordinary RT-PCR by 1000 folds, and the inter-assay and intra-assay CV values were all less than 0.99%. The newly developed RT-qPCR assay exhibited good sensitivity and reproducibility, which will provide technical support for the reliable and specific rapid diagnosis, and quantitative analysis of GETV infection.

Development of a TaqMan probe-based quantitative reverse transcription PCR assay for detection of Getah virus RNA.

Getah virus (GETV), a mosquito-borne virus that mainly infects horses and pigs, has emerged and spread in China. We developed a highly specific and reproducible TaqMan probe-based quantitative reverse transcription PCR (RT-qPCR) assay targeting the non-structural protein 1 of GETV, whose detection limit is 25.5 copies/µL, which is 100-fold higher than that of conventional RT-PCR. RT-qPCR was used to detect GETV RNA in mosquito and animal clinical samples, showing that the accuracy of RT-qPCR was higher than that of conventional RT-PCR. The newly developed RT-qPCR assay may be a useful alternative tool for rapid, simple and specific diagnosis of GETV infection.

Development of a real-time reverse transcription PCR assay for detection of a novel nidovirus associated with a neurological disease of the Australian brushtail possum (Trichosurus vulpecula)

AIMS: To develop a quantitative reverse transcription PCR (RT-qPCR) assay for detection of the putative wobbly possum disease (WPD) virus and to apply this test to investigate the viral load in archival tissues from past WPD transmission studies. METHODS: The real-time assay was developed as a two-step RT-qPCR in a SYBR green format and validated using serial dilutions of a linearised plasmid containing target DNA. The copy number values were normalised to the amount of RNA in each reverse transcription reaction and presented as viral copies/pg RNA. The viral load was determined in archival samples from animals that had received inoculations of infectious WPD tissue suspensions. Thirty samples originating from 22 possums, comprising five samples from three healthy possums and 25 samples from 19 possums that had received inoculations of infectious WPD tissue suspensions were tested. RESULTS: The assay was linear (R2 > 0.99) within the tested range from 1 to 107 target copies/µL, with an efficiency of >90%. The intra-assay variability CV values ranged from 0.8 to 4.5% for different standards, with the inter-assay variability CV values ranging from 0.4 to 2.5%, indicating good precision and reproducibility of the assay. The novel nidovirus was detected in all 25 samples from WPD-affected possums. Tissues from three control possums and from one experimentally infected rabbit were negative for WPD RNA. The viral load in WPD-positive tissues differed between individual possums and between tissue types, ranging from 2.2 to 359,980 copies/pg RNA. The highest viral load was detected in liver, followed by brain, spleen, kidney and urine. There was a more than four log difference in the viral load between pools of tissues originating from two outbreaks of WPD in different geographical regions. CONCLUSIONS: Detection of viral RNA in a variety of tissues from WPD affected possums, including brain, is consistent with the multi-organ distribution of histopathological lesions observed in WPD. Our data suggest that liver may constitute the sample of choice for diagnostic testing. Differences in the viral load in tissues from possums inoculated with infectious WPD tissue suspensions from Rotorua or Invermay origin suggest that WPD viruses with different biological properties may exist. CLINICAL RELEVANCE: We have developed a RT-qPCR assay for detection of the putative WPD virus. The test showed good sensitivity and reproducibility over the wide dynamic range of template concentrations. It provides a tool for future diagnostic and research purposes.