Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus

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 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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Comparison of nine different commercially available molecular assays for detection of SARS-CoV-2 RNA

European Journal of Clinical Microbiology & Infectious Diseases ◽

10.1007/s10096-021-04159-9 ◽

2021 ◽

Author(s):

Ute Eberle ◽

◽

Clara Wimmer ◽

Ingrid Huber ◽

Antonie Neubauer-Juric ◽

...

Keyword(s):

Real Time ◽

Rt Pcr ◽

Diagnostic Assays ◽

Molecular Assays ◽

Pcr Assays ◽

Laboratory Experiences

AbstractTo face the COVID-19 pandemic, the need for fast and reliable diagnostic assays for the detection of SARS-CoV-2 is immense. We describe our laboratory experiences evaluating nine commercially available real-time RT-PCR assays. We found that assays differed considerably in performance and validation before routine use is mandatory.

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Quantitation of replication of the HCV genome in human livers with end-stage cirrhosis by strand-specific real-time RT-PCR assays: Methods and clinical relevance

Journal of Medical Virology ◽

10.1002/jmv.21510 ◽

2009 ◽

Vol 81 (9) ◽

pp. 1569-1575 ◽

Cited By ~ 4

Author(s):

Lan Lin ◽

Louis Libbrecht ◽

Jannick Verbeeck ◽

Chris Verslype ◽

Tania Roskams ◽

...

Keyword(s):

Real Time ◽

Clinical Relevance ◽

Rt Pcr ◽

Pcr Assays ◽

Human Livers ◽

End Stage

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Development of Reverse Transcription (RT)-PCR and Real-Time RT-PCR Assays for Rapid Detection and Quantification of Viable Yeasts and Molds Contaminating Yogurts and Pasteurized Food Products

Applied and Environmental Microbiology ◽

10.1128/aem.69.7.4116-4122.2003 ◽

2003 ◽

Vol 69 (7) ◽

pp. 4116-4122 ◽

Cited By ~ 106

Author(s):

Gianluca Bleve ◽

Lucia Rizzotti ◽

Franco Dellaglio ◽

Sandra Torriani

Keyword(s):

Real Time ◽

Food Products ◽

Fungal Species ◽

Universal Primers ◽

Rt Pcr ◽

Specificity And Sensitivity ◽

Actin Mrna ◽

Pcr Assays ◽

Food Commodities ◽

Yeasts And Molds

ABSTRACT Reverse transcriptase PCR (RT-PCR) and real-time RT-PCR assays have been used to detect and quantify actin mRNA from yeasts and molds. Universal primers were designed based on the available fungal actin sequences, and by RT-PCR they amplified a specific 353-bp fragment from fungal species involved in food spoilage. From experiments on heat-treated cells, actin mRNA was a good indicator of cell viability: viable cells and cells in a nonculturable state were detected, while no signal was observed from dead cells. The optimized RT-PCR assay was able to detect 10 CFU of fungi ml−1 in pure culture and 103 and 102 CFU ml−1 in artificially contaminated yogurts and pasteurized fruit-derived products, respectively. Real-time RT-PCR, performed on a range of spoiled commercial food products, validated the suitability of actin mRNA detection for the quantification of naturally contaminating fungi. The specificity and sensitivity of the procedure, combined with its speed, its reliability, and the potential automation of the technique, offer several advantages to routine analysis programs that assess the presence and viability of fungi in food commodities.

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Accurate and precise real-time RT-PCR assays for the identification of astrovirus associated encephalitis in cattle

Scientific Reports ◽

10.1038/s41598-018-27533-8 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 6

Author(s):

Ramona Lüthi ◽

Céline L. Boujon ◽

Ronja Kauer ◽

Michel C. Koch ◽

Ilias G. Bouzalas ◽

...

Keyword(s):

Real Time ◽

Rt Pcr ◽

Pcr Assays

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A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.00505-18 ◽

2018 ◽

Vol 56 (8) ◽

Cited By ~ 3

Author(s):

Nawal El Houmami ◽

Guillaume André Durand ◽

Janek Bzdrenga ◽

Anne Darmon ◽

Philippe Minodier ◽

...

Keyword(s):

Real Time ◽

Malate Dehydrogenase ◽

Real Time Pcr ◽

Detection Sensitivity ◽

Rt Pcr ◽

Pcr Assay ◽

Content Type ◽

Clinical Specimens ◽

Specificity And Sensitivity ◽

Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

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Development and Evaluation of Novel and Highly Sensitive Single-Tube Nested Real-Time RT-PCR Assays for SARS-CoV-2 Detection

International Journal of Molecular Sciences ◽

10.3390/ijms21165674 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5674

Author(s):

Cyril Chik-Yan Yip ◽

Siddharth Sridhar ◽

Kit-Hang Leung ◽

Anthony Chin-Ki Ng ◽

Kwok-Hung Chan ◽

...

Keyword(s):

Real Time ◽

Limit Of Detection ◽

Rt Pcr ◽

Single Tube ◽

Highly Sensitive ◽

Testing Algorithms ◽

Pcr Assays ◽

Human Coronaviruses ◽

Pooled Sample

Sensitive molecular assays are critical for coronavirus disease 2019 (COVID-19) diagnosis. Here, we designed and evaluated two single-tube nested (STN) real-time RT-PCR assays, targeting SARS-CoV-2 RdRp/Hel and N genes. Both STN assays had a low limit of detection and did not cross react with other human coronaviruses and respiratory viruses. Using 213 initial respiratory specimens from suspected COVID-19 patients, the sensitivity of both the STN COVID-19-RdRp/Hel and the STN COVID-19-N assays was 100% (99/99), while that of the comparator non-nested N assay was 95% (94/99). Among 108 follow-up specimens from confirmed COVID-19 patients who tested negative by the non-nested COVID-19-RdRp/Hel assay, 28 (25.9%) were positive for SARS-CoV-2 by the STN COVID-19-RdRp/Hel or the STN COVID-19-N assay. To evaluate the performance of our novel STN assays in pooled specimens, we created four sample pools, with each pool consisting of one low positive specimen and 49 negative specimens. While the non-nested COVID-19-RdRp/Hel assay was positive in only one of four sample pools (25%), both of the STN assays were positive in two of four samples pools (50%). In conclusion, the STN assays are highly sensitive and specific for SARS-CoV-2 detection. Their boosted sensitivity offers advantages in non-traditional COVID-19 testing algorithms such as saliva screening and pooled sample screening during massive screening.

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