Development and application of a low-priced duplex quantitative PCR assay based on SYBR Green I for the simultaneous detection of porcine deltacoronavirus and porcine sapelovirus
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ABSTRACT:
SUBMITTER: Lu S
PROVIDER: S-EPMC10581527 | biostudies-literature | 2023 Mar
REPOSITORIES: biostudies-literature
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Project description:The development of a rapid, specific, and sensitive SYBR Green I-based duplex real-time quantitative PCR assay is described for the simultaneous detection of porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 3 (PCV3). The assay specifically detected PEDV and PCV3, with no fluorescence detected for other non-targeted pig pathogens. The assay showed a good linear relationship, and the limits of detection for this assay were 34.6 copies/?L and 61.2 copies/?L for PEDV and PCV3, respectively. The assay exhibited high repeatability and reproducibility, with intra-assay and inter-assay variation coefficients less than 2.0%. A clinical evaluation using intestinal tissue and fecal samples from piglets suffering from diarrhea at different pig farms in China revealed that the singular infection rates of PEDV and PCV3 were 43.94% (29/66) and 16.67% (11/66), respectively, while the co-infection rate of PCV3 with PEDV was 27.27% (18/66). The results indicate this assay is a rapid and reliable diagnostic tool for PEDV and PCV3 monitoring and surveillance in the field, and provides technical support for the quantitative detection of clinical samples infected or co-infected with PEDV and PCV3.
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Project description:Porcine circoviruses (PCV) include PCV1, PCV2, and the new-emerging PCV3. PCV2 is pathogenic to pigs, but the pathogenicity of PCV3 in pigs is debatable. Recently, there have been frequent reports of PCV2 and PCV3 co-infections in clinical samples. Thus, it would be practical to develop a duplex PCR method to detect PCV2 and PCV3 simultaneously. In this study, specific primers and probes were designed to target PCV2 cap and PCV3 rep genes. A duplex real-time PCR method was then developed to detect the two viruses. The assay was found to be highly specific, sensitive, and reproducible for PCV2/3 without cross-reactions with other swine pathogens. The sensitivity of this assay was 2.9 copies for the PCV2 plasmid and 22.5 copies for the PCV3 plasmid. The established assay was then used to detect PCV2/3 infection in 340 clinical samples collected in the first half of 2017. The results showed that the co-infection rate of PCV2/3 in the samples was 27.6%. Our study provides an important tool that can be used to perform urgently needed surveys for the two porcine circoviruses to evaluate their impact on the swine industry.
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Project description:BACKGROUND: Co-infections of Apple chlorotic leaf spot virus (ACLSV) and Cherry green ring mottle virus (CGRMV) in peach is common in China and have resulted in significant yield reductions. A reliable, sensitive and quantitive method is needed to detect and distinguish between ACLSV and CGRMV in peach. FINDINGS: We developed a sensitive and specific SYBR Green-I based RT-qPCR for the quantification of ACLSV and CGRMV in different peach tissues, and a duplex RT-qPCR system to detect ACLSV and CGRMV simultaneously. The RT-qPCR method was optimized using standard samples transcribed by the T7 Large Scale RNA Production System in vitro. The peach genes, RNA Polymerase subunit II (RPII) and Ubiquitin 10 (UBQ10), which were used as the internal controls for the quantification assay also showed good expression stability in this system. Single RT-qPCR assays showed that CGRMV in peach accumulates to a higher level than ACLSV. The detection limits of the duplex RT-qPCR assay were 10² and 10? copies for ACLSV and CGRMV, respectively. The sensitivity of the duplex RT-qPCR was as high as RT-qPCR and higher than RT-PCR. CONCLUSIONS: The SYBR Green-I RT-qPCR assay provided a sensitive, specific and reliable method for the detection and quantification of ACLSV and CGRMV in different peach tissues. The duplex RT-qPCR system provided a sensitive and specific method to detect and differentiate between ACLSV and CGRMV in a single sample. This RT-qPCR assay could be a useful tool for the routine diagnosis of these two viruses and for disease epidemiology studies in peach orchards.
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Project description:BackgroundWaterfowl parvoviruses, including goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV), can cause seriously diseases in geese and ducks. Developing a fast and precise diagnosis assay for these two parvoviruses is particularly important.ResultsA duplex SYBR Green I-based quantitative real-time PCR assay was developed for the simultaneous detection and differentiation of GPV and MDPV. The assay yielded melting curves with specific single peak (Tm = 87.3 ± 0.26 °C or Tm = 85.4 ± 0.23 °C) when GPV or MDPV was evaluated, respectively. When both parvoviruses were assessed in one reaction, melting curves with specific double peaks were yielded.ConclusionThis duplex quantitative RT-PCR can be used to rapid identify of GPV and MDPV in field cases and artificial trials, which make it a powerful tool for diagnosing, preventing and controlling waterfowl parvovirus infections.
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Project description:The emergence in Japan of field isolates of type 1 porcine reproductive and respiratory syndrome virus (PRRSV) suggests problems with control. We therefore developed a one-step real-time reverse transcription polymerase chain reaction (qRT-PCR) with improved sensitivity that detects as little as 1 × 10(-2) TCID50/ml of viral RNA. We tested serum samples collected in January and September 2008, October 2009 and January 2011 from a farm with an outbreak and found infected pigs between January and September 2008, but not in January 2011. Further, between 2008 and 2011, we did not detect infection in pigs at 8 nearby farms or in 2,052 serum samples collected from pigs from 74 farms in 12 prefectures. This assay should help prevent future outbreaks.
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Project description:Since both feline parvovirus (FPV) and feline bocavirus (FBoV) can cause diarrhea in cats, it is difficult to distinguish them clinically. This study aimed to develop a SYBR Green I-based duplex real-time polymerase chain reaction (PCR) assay for distinguishing FPV and FBoV-1 on the basis of the melting temperature of the PCR product. A total of 132 fecal samples from different domestic and feral cats were collected, and the results of SYBR Green I-based duplex real-time PCR assay were compared with those of the traditional PCR assay for a comprehensive evaluation. The melting temperatures were found to be 86 °C and 77.5 °C for FBoV-1 and FPV, respectively, and no specific melting peaks for other non-targeted feline viruses were observed. The data obtained from this assay had a good linear relationship; the detection limits of FPV and FBoV-1 were 2.907 × 101 copies/μL and 3.836 × 101 copies/μL, respectively. In addition, the experiment exhibited high reproducibility. The positive detection rates of the SYBR Green I-based duplex real-time PCR assay for FPV and FBoV-1 were 16.67% (22/132) and 6.82% (9/132), respectively, and the positive detection rate for co-infection with FPV and FBoV-1 was 3.03% (4/132). This result was much more sensitive than that of the traditional PCR method. Thus, the developed SYBR Green I-based assay is a sensitive, rapid, specific, and reliable method for the clinical diagnosis of FPV and FBoV-1 and can provide technical support for the simultaneous detection of co-infection with these viruses in the future.Supplementary informationThe online version contains supplementary material available at 10.1007/s13205-021-02947-w.
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Project description:Hantaan virus (HTNV) is a major zoonotic pathogen that causes hemorrhagic fever with renal syndrome (HFRS) in Asia, especially in China. Shaanxi province, which is located in northwest of China, is one of the areas in China most severely afflicted with HFRS epidemics annually. This study aims to establish a quantitative RT-PCR (qRT-PCR) assay to detect HTNV both in cell culture and clinical serum samples. We established a SYBR Green I-based one-step qRT-PCR assay that targets the S segment of the HTNV genome for rapid detection and quantification. The HTNV cRNA standards were constructed by in vitro transcription, and the copy numbers of the HTNV cRNA were quantified. Standard curve was generated by determining the mean cycle threshold (Ct) values versus 10-fold serial dilutions of the HTNV cRNA over a range of 1 × 10(8) to 1 × 10(3) copies/μl. The standard curve had a reaction efficiency of 102.1%, a correlation coefficient (R(2)) of 0.998, and a slope of -3.273. The coefficient of variation (CV) of the intra- and inter-assays ranged from 0.68% to 3.00% and from 0.86% to 3.21%, respectively. The cycle intervals of the qRT-PCR assay between each dilution ranged from 2.9 to 3.8 cycles, and the lowest detection limit of the qRT-PCR assay was 10 copies/μl. The assay exhibited high specificity that was confirmed by melting curve analysis, and no cross reaction with the Seoul virus (SEOV) and other viruses (HBV, HCV and HIV) was observed. HTNV RNA was also detected in the 27 serum samples of clinical HFRS patients using the assay, and the HTNV RNA viral load ranged from 2.06 × 10(1) to 1.95 × 10(5) copies/μl. The SYBR Green I-based one-step qRT-PCR assay is a sensitive, specific, reproducible, and simple method for detecting and quantifying HTNV in cell culture and clinical samples.
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