Simultaneous Detection and Differentiation of Highly Virulent and Classical Chinese-Type Isolation of PRRSV by Real-Time RT-PCR

Porcine reproductive and respiratory syndrome (PRRS) is a leading disease in pig industry worldwide and can result in serious economic losses each year. The PRRS epidemic situation in China has been very complicated since the unprecedented large-scale highly pathogenic PRRS (HP-PRRS) outbreaks in 2006. And now the HP-PRRS virus (HP-PRRSV) and classical North American type PRRSV strains have coexisted in China. Rapid differential detection of the two strains of PRRSV is very important for effective PRRS control. The real-time RT-PCR for simultaneous detection and differentiation of HP-PRRSV and PRRSV by using both SYBR Green and TaqMan probes was developed and validated. Both assays can be used for rapid detection and strain-specific identification of HP-PRRSV and PRRSV. However, the TaqMan probe method had the highest detection rate whereas the conventional RT-PCR was the lowest. The real-time RT-PCR developed based on SYBR Green and TaqMan probe could be used for simultaneous detection and differentiation of HP-PRRSV and PRRSV in China, which will benefit much the PRRS control and research.

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Sensitive SYBR Green—Real Time PCR for the Detection and Quantitation of Avian Rotavirus A

Veterinary Sciences ◽

10.3390/vetsci6010002 ◽

2018 ◽

Vol 6 (1) ◽

pp. 2 ◽

Cited By ~ 2

Author(s):

David De la Torre ◽

Claudete Astolfi-Ferreira ◽

Ruy Chacon ◽

Antonio Piantino Ferreira

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Sybr Green ◽

Molecular Techniques ◽

Economic Losses ◽

Rt Pcr ◽

Chain Reaction ◽

Rotavirus A ◽

Avian Nephritis Virus ◽

Polymerase Chain

Avian rotavirus A (ARtV-A) is a virus that affects young birds, causing acute diarrhea and economic losses in the poultry industry worldwide. The techniques used for the diagnosis of ARtV-A include electron microscopy, isolation in cell culture, and serology, as well as molecular techniques, such as the reverse transcription-polymerase chain reaction (RT-PCR). The objective of this work was to standardize a real-time RT-polymerase chain reaction (RT-qPCR) using SYBR Green chemistry for the rapid detection and quantification of ARtV-A from bird tissues and materials fixed on FTA cards on the basis of the nucleotide sequence of segment 6 (S6), which codes for the structural VP6 protein of ARtV-A. The results show the efficient amplification of the proposed target, with a limit of detection (LoD) of one copy gene (CG) per microliter of cDNA and a limit of quantification (LoQ) of 10 CGs per microliter. The efficiency of the primers was determined to be 95.66% using a standard curve, with an R2 value of 0.999 and a slope of −3.43. The specificity was determined using samples coinfected with ARtV-A, the chicken parvovirus, the chicken astrovirus, and the avian nephritis virus as positive controls and commercially available vaccines of the infectious bronchitis virus, infectious bursa disease virus, avian reovirus and healthy organs as negative controls. This technique, which lacks nonspecific PCR products and dimers, demonstrated greater sensitivity and specificity than conventional RT-PCR, and it reduced the analysis time by more than 50%.

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Simultaneous detection and genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCR and amplicon melting curve analysis using SYBR Green

Research in Veterinary Science ◽

10.1016/j.rvsc.2007.10.003 ◽

2008 ◽

Vol 85 (1) ◽

pp. 184-193 ◽

Cited By ~ 24

Author(s):

E. Martínez ◽

P. Riera ◽

M. Sitjà ◽

Y. Fang ◽

S. Oliveira ◽

...

Keyword(s):

Real Time ◽

Simultaneous Detection ◽

Melting Curve ◽

Curve Analysis ◽

Sybr Green ◽

Respiratory Syndrome Virus ◽

Melting Curve Analysis ◽

Rt Pcr

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Genetic Diversity of Porcine Reproductive and Respiratory Syndrome Virus and Evaluation of Three One-Step Real-Time RT-PCR Assays in Korea

10.21203/rs.3.rs-1149203/v1 ◽

2021 ◽

Author(s):

Go-Eun Shin ◽

Ji-Young Park ◽

Kyoung-Ki Lee ◽

Mi-Kyeong Ko ◽

Bok-Kyung Ku ◽

...

Keyword(s):

Amino Acid ◽

Sequence Analysis ◽

Real Time ◽

Economic Losses ◽

Respiratory Syndrome Virus ◽

Nucleotide Sequence Analysis ◽

Rt Pcr ◽

The Real ◽

One Step ◽

Pcr Assays

Abstract Background Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses in the global swine industry. Frequent genetic variations in this virus cause difficulties in controlling and accurately diagnosing PRRSV. Methods In this study, we investigated the genetic characteristics of PRRSV-1 and PRRSV-2 circulating in Korea from January 2018 to September 2021 and evaluated three one-step real-time reverse transcription polymerase chain reaction (RT-PCR) assays. Results A total of 129 lung samples were collected, consisting of 47 samples for PRRSV-1, 62 samples for PRRSV-2, and 20 PRRSV-negative samples. Nucleotide sequence analysis of open reading frames (ORFs) 5, ORF6, and ORF7 genes from PRRSV samples showed that PRRSV-1 belonged to subgroup A (43/47, 91.49%) and subgroup C (4/47, 8.51%), whereas PRRSV-2 was classified as lineage 1 (25/62, 40.32%), Korean lineage (Kor) C (13/62, 20.97%), Kor B (10/62, 16.13%), lineage 5 (9/62, 14.52%), and Kor A (5/62, 8.06%). Amino acid sequence analysis showed that the neutralizing epitope and T cell epitope of PRRSV-1, and the decoy epitope region and hypervariable regions of PRRSV-2 had evolved under positive selection pressure. In particular, the key amino acid substitutions were found at positions 102 and 104 of glycoprotein 5 (GP5) in some PRRSV-2, and at positions 10 and 70 of membrane protein (M) in most PRRSV-2. In addition, one-step real-time RT-PCR assays, comprising two commercial tests and one test recommended by the World Organization for Animal Health (OIE), were evaluated. Conclusion The results revealed that two of the real-time RT-PCR assays had high sensitivities and specificities, whereas the real-time RT-PCR assay of the OIE had low sensitivity due to mismatches between nucleotides of Korean PRRSVs and forward primers. In this study, we genetically characterized recent PRRSV occurrences and evaluated three one-step real-time RT-PCR assays used in Korea.

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Simultaneous detection of HCV and HIV-1 by SYBR Green real time multiplex RT-PCR technique in plasma samples

Molecular and Cellular Probes ◽

10.1016/j.mcp.2005.12.005 ◽

2006 ◽

Vol 20 (3-4) ◽

pp. 223-229 ◽

Cited By ~ 22

Author(s):

Davide Gibellini ◽

Federica Gardini ◽

Francesca Vitone ◽

Pasqua Schiavone ◽

Giuliano Furlini ◽

...

Keyword(s):

Real Time ◽

Simultaneous Detection ◽

Sybr Green ◽

Plasma Samples ◽

Rt Pcr ◽

Hiv 1

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Clinical Evaluation of a Combo Rapid Antigen Test QuickNavi-Flu+COVID19 Ag for Simultaneous Detection of SARS-CoV-2 and Influenza Viruses

10.1101/2021.12.05.21267215 ◽

2021 ◽

Author(s):

Yuto Takeuchi ◽

Yusaku Akashi ◽

Yoshihiko Kiyasu ◽

Norihiko Terada ◽

Yoko Kurihara ◽

...

Keyword(s):

Real Time ◽

Sensitivity And Specificity ◽

Clinical Performance ◽

Simultaneous Detection ◽

Influenza Viruses ◽

Antigen Test ◽

Sample Collection ◽

Rt Pcr ◽

The Real ◽

Reverse Transcription Pcr

AbstractIntroductionSince respiratory sample collection is an uncomfortable experience, simultaneous detection of pathogens with a single swab is preferable. We prospectively evaluated the clinical performance of a newly developed antigen test QuickNavi-Flu+COVID19 Ag (Denka Co., Ltd., Tokyo, Japan) which can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses at the same time with a single testing device.MethodsIncluded were those who were suspected of contracting coronavirus disease 2019 (COVID-19) and referred to a PCR center at Ibaraki prefecture in Japan, between August 2, 2021 to September 13, 2021, when the L452R mutant strains of SARS-CoV-2 were prevalent. Additional nasopharyngeal samples and anterior nasal samples were obtained for the antigen test and were compared with a reference reverse transcription PCR (RT-PCR) using nasopharyngeal samples.ResultsIn total, 1510 nasopharyngeal samples and 862 anterior nasal samples were evaluated. For SARS-CoV-2 detection in nasopharyngeal samples, the sensitivity and specificity of the antigen test were 80.9% and 99.8%, respectively. The sensitivity and specificity using anterior nasal samples were 67.8% and 100%, respectively. In symptomatic cases, the sensitivities increased to 88.3% with nasopharyngeal samples and 73.7% with anterior nasal samples. There were three cases of discrepant results between the antigen test and the real-time RT-PCR. All of them were positive with the antigen test but negative with the real-time RT-PCR in SARS-CoV-2 detection. During the study period, influenza viruses were not detected.ConclusionA combo kit, QuickNavi-Flu+COVID19 Ag, showed an acceptable sensitivity and sufficient specificity for SARS-CoV-2 detection, especially using nasopharyngeal sample collected from symptomatic patients.

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Development and Evaluation of a SYBR Green–Based Real-Time Multiplex RT-PCR Assay for Simultaneous Detection and Serotyping of Dengue and Chikungunya Viruses

Journal of Molecular Diagnostics ◽

10.1016/j.jmoldx.2015.06.008 ◽

2015 ◽

Vol 17 (6) ◽

pp. 722-728 ◽

Cited By ~ 14

Author(s):

Huixin Chen ◽

Mariya Parimelalagan ◽

Yee Ling Lai ◽

Kim Sung Lee ◽

Evelyn Siew-Chuan Koay ◽

...

Keyword(s):

Real Time ◽

Simultaneous Detection ◽

Sybr Green ◽

Rt Pcr ◽

Pcr Assay

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A duplex SYBR Green I-based real-time RT-PCR assay for the simultaneous detection and differentiation of Massachusetts and non-Massachusetts serotypes of infectious bronchitis virus

Molecular and Cellular Probes ◽

10.1016/j.mcp.2013.06.001 ◽

2013 ◽

Vol 27 (5-6) ◽

pp. 184-192 ◽

Cited By ~ 19

Author(s):

Ana M. Acevedo ◽

Carmen L. Perera ◽

Armando Vega ◽

Liliam Ríos ◽

Liani Coronado ◽

...

Keyword(s):

Real Time ◽

Infectious Bronchitis Virus ◽

Simultaneous Detection ◽

Sybr Green ◽

Sybr Green I ◽

Rt Pcr ◽

Pcr Assay ◽

Infectious Bronchitis

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A New Multiplex Real-Time RT-PCR for Simultaneous Detection and Differentiation of Avian Bornaviruses

Viruses ◽

10.3390/v13071358 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1358

Author(s):

Brigitte Sigrist ◽

Jessica Geers ◽

Sarah Albini ◽

Dennis Rubbenstroth ◽

Nina Wolfrum

Keyword(s):

Real Time ◽

Simultaneous Detection ◽

Epidemiological Studies ◽

Virus Species ◽

Rt Pcr ◽

P Gene ◽

Field Samples ◽

Causative Agents ◽

Species Specific ◽

Proventricular Dilatation Disease

Avian bornaviruses were first described in 2008 as the causative agents of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly diverse avian bornaviruses covering at least five viral species have been discovered in different bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays, allowing the detection of one particular virus, or of assays with a broad detection spectrum, which, however, do not allow for the simultaneous specification of the detected virus. To facilitate the simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were designed to recognize conserved regions within the overlapping X/P gene and probes were selected to detect virus species-specific regions within the target region. The optimization of the assay resulted in the sensitive and specific detection of bornaviruses of Psittaciformes, Passeriformes, and aquatic birds. Finally, the new rRT-PCR was successfully employed to detect avian bornaviruses in field samples from various avian species. This assay will serve as powerful tool in epidemiological studies and will improve avian bornavirus detection.

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