Detection and Typing of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus by Multiplex Real-Time RT-PCR

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases to swine industry worldwide. Due to the heterogeneity of field isolates, accurate detection of the PRRS virus is a diagnostic challenge. Recently, co-infection with NA-PRRSV, EU-PRRSV and HP-PRRSV isolates continuously increases in many countries, resulting in a significant impact on PRRSV diagnostics and disease control on farms. To facilitate rapid diagnosis and reliable discrimination of NA-PRRSV, EU-PRRSV and HP-PRRSV, a multiplex RT-PCR assay was established with three pairs of primers targeting highly conservative regions of nsp2 gene with predicted multiplex RT-PCR products of 364 bp, 161 bp and 259 bp, respectively. The primer pairs were optimized to be highly specific for PRRSV genotypes and were able to detect the target gene at the limit of 102 copies/μL for each gene. Clinical samples were used to evaluate this multiplex RT-PCR in parallel with a commercial real-time RT-PCR kit. Results showed over 95.2% (20/21 samples) agreement between the mRT-PCR and the real-time RT-PCR kit. Hence, it indicated that this multiplex RT-PCR could be useful for rapid and deferential diagnosis of NA-PRRSV, EU-PRRSV and HP-PRRSV in swine farms.

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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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The development of a rapid SYBR one step real-time RT-PCR for detection of porcine reproductive and respiratory syndrome virus

Virology Journal ◽

10.1186/1743-422x-7-90 ◽

2010 ◽

Vol 7 (1) ◽

pp. 90 ◽

Cited By ~ 12

Author(s):

Hong Tian ◽

JingYan Wu ◽

YouJun Shang ◽

Yan Cheng ◽

XiangTao Liu

Keyword(s):

Real Time ◽

Respiratory Syndrome Virus ◽

Rt Pcr ◽

One Step

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Simultaneous Detection of North American and European Porcine Reproductive and Respiratory Syndrome Virus Using Real-Time Quantitative Reverse Transcriptase–PCR

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870501700211 ◽

2005 ◽

Vol 17 (2) ◽

pp. 165-170 ◽

Cited By ~ 57

Author(s):

Steven B. Kleiboeker ◽

Susan K. Schommer ◽

Sang-Myeong Lee ◽

Sandy Watkins ◽

Wayne Chittick ◽

...

Keyword(s):

Reverse Transcriptase ◽

Real Time ◽

North American ◽

Simultaneous Detection ◽

Viral Rna ◽

Respiratory Syndrome Virus ◽

Analytical Sensitivity ◽

Rt Pcr ◽

Field Isolates ◽

Pcr Assays

Porcine reproductive and respiratory syndrome (PRRS) is 1 of the most economically important diseases of swine. Detection of the etiologic agent, PRRS virus (PRRSV), represents a diagnostic challenge due to the heterogeneity of field isolates as well as the propensity for swine to develop persistent infection in which virus is difficult to detect. Recently European (EU) lineage PRRSV isolates, which are genetically divergent from North American (NA) isolates, have been introduced into NA swine further complicating efforts to diagnose this disease. In this study, real-time ( TaqMan) reverse transcriptase (RT)–PCR assays were developed for multiplex detection, differentiation, and quantification of NA and EU PRRSV field isolates. Oligonucleotide primers and dual-labeled probes were selected from conserved regions of open-reading frame 7 and the 3'-untranslated region. The real-time RT-PCR assays described for the NA or EU genotype of PRRSV detected viral RNA from 83/83 strains (74 NA; 9 EU) previously isolated by cell culture between 1992 and 2003. The analytical sensitivity of both assays was consistently found to be less than a single TCID50, which corresponded to 5–10 RNA molecules, and was not significantly reduced when the reactions were performed in a multiplex format. When performing multiplex reactions, sensitive detection was possible even when 1 viral RNA concentration was up to 5,000-fold higher than the second. The diagnostic sensitivity and specificity of the multiplex reaction was found to be at a minimum equivalent to that of both nested RT-PCR and virus isolation.

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Deteksi Antigen Virus Avian Influenza pada Ayam Kampung di Pasar Hewan Beringkit dan Pasar Umum Galiran, Bali

Indonesia Medicus Veterinus ◽

10.19087/imv.2020.9.5.757 ◽

2020 ◽

Vol 9 (5) ◽

pp. 757-772

Author(s):

Annisa Musdalifa ◽

Gusti Ayu Yuniati Kencana ◽

I Nyoman Suartha

Keyword(s):

Polymerase Chain Reaction ◽

Avian Influenza ◽

Real Time ◽

Highly Pathogenic Avian Influenza ◽

Rt Pcr ◽

Chain Reaction ◽

Chi Square ◽

Highly Pathogenic ◽

Pathogenic Avian Influenza ◽

Polymerase Chain

Avian Infuenza termasuk ke dalam kelompok penyakit menular strategis dan bersifat zoonosis. Penyebabnya adalah virus Highly Pathogenic Avian Influenza (HPAI) subtipe H5N1. Introduksi virus Avian Infuenza subtipe H5N1 dapat melalui jalur pasar karena berpotensi dalam penyebarannya penyakit Avian Influenza pada ayam kampung secara alamiah melalui kontak langsung dengan unggas lain. Penelitian ini dilakukan di Pasar Hewan Beringkit, Kabupaten Badung dan Pasar Umum Galiran, Kabupaten Klungkung. Tujuan penelitian ini untuk mengetahui keberadaan virus Avian Influenza subtipe H5N1 pada ayam kampung di Pasar Hewan Beringkit dan Pasar Umum Galiran. Sampel yang digunakan sebanyak 120 sampel swab kloaka dan swab trakea ayam kampung, dengan jumlah masing-masing pasar sebanyak 60 sampel. Periode pengambilan sampel dilakukan selama dua bulan dengan periode pengambilan setiap dua minggu sekali sebanyak empat kali. Pengambilan sampel dilakukan pada ayam kampung berumur di atas tiga bulan yang tidak divaksinasi. Isolasi virus pada telur ayam berembrio (TAB) berumur sembilan hari dan identifikasinya dengan uji hemaglutinasi (HA), uji hambatan hemaglutinasi (HI) serta Real Time Polymerase Chain Reaction (RT-PCR). Analisis data hasil uji antigen Avian Influenza dengan uji statistika non-parametrik Chi-square (?2). Hasil uji sampel swab kloaka dan trakea ayam kampung asal Pasar Beringkit positif virus Avian Influenza sebesar 5%, sedangkan sampel positif virus Avian Influenza asal Pasar Galiran sebesar 6,7%. Berdasarkan hasil penelitian maka dapat disimpulkan bahwa virus Avian Influenza terdeteksi pada ayam kampung di Pasar Hewan Beringkit dan Pasar Umum Galiran, virus AI masih bersikulasi pada daerah asal peternakan dan penyakit AI masih mewabah di Bali.

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