scholarly journals Multiplex RT-PCR assay to differentiate genotypes of porcine reproductive and respiratory syndrome virus in swine

2019 ◽  
Vol 18 (06) ◽  
pp. 8-13
Author(s):  
Phat X. Dinh
Keyword(s):  
Real Time ◽  
Clinical Samples ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Swine Industry ◽  
Diagnostic Challenge ◽  
Nsp2 Gene ◽  
Prrs Virus ◽  
Pcr Products

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.

scholarly journals Description and Validation of a Novel Real-Time RT-PCR Enterovirus Assay

2008 ◽  
Vol 54 (2) ◽  
pp. 406-413 ◽  
Author(s):  
Weston C Hymas ◽  
Wade K Aldous ◽  
Edward W Taggart ◽  
Jeffery B Stevenson ◽  
David R Hillyard
Keyword(s):  
Sequence Analysis ◽  
Single Nucleotide Polymorphisms ◽  
Real Time ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Nucleotide ◽  
The Real ◽  
Nontranslated Region

Abstract Background: Enteroviruses are a leading cause of aseptic meningitis in adult and pediatric populations. We describe the development of a real-time RT-PCR assay that amplifies a small target in the 5′ nontranslated region upstream of the classical Rotbart enterovirus amplicon. The assay includes an RNA internal control and incorporates modified nucleotide chemistry. Methods: We evaluated the performance characteristics of this design and performed blinded parallel testing on clinical samples, comparing the results with a commercially available RT-PCR assay (Pan-Enterovirus OligoDetect kit) that uses an enzyme immunoassay–like plate end detection. Results: We tested 778 samples and found 14 discrepant samples between the 2 assays. Of these, the real-time assay detected 6 samples that were negative by the OligoDetect kit, 5 of which were confirmed as positive by sequence analysis using an alternative primer set. Eight discrepant samples were positive by the OligoDetect kit and real-time negative, with 6 confirmed by sequencing. Overall, detection rates of 97% and 96% were obtained for the OligoDetect kit and real-time assays, respectively. Sequence analysis revealed the presence of a number of single nucleotide polymorphisms in the targeted region. The comparative sensitivities of the 2 assays were equivalent, with the limit of detection for the real-time assay determined to be approximately 430 copies per milliliter in cerebrospinal fluid. Conclusions: This novel real-time enterovirus assay is a sensitive and suitable assay for routine clinical testing. The presence of single nucleotide polymorphisms can affect real-time PCR assays.

scholarly journals Systematic Review and Meta-analysis of Real-time Polymerase Chain Reaction assay for the detection of COVID-19 from clinical samples in low-and middle-income countries: Protocol

2021 ◽  
Author(s):  
Emmanuel Oladipo Babafemi
Keyword(s):  
Systematic Review ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Meta Analysis ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Middle Income ◽  
Polymerase Chain

Abstract Background: COVID-19 has spread globally since its discovery in Hubei province, China in December 2019 and became pandemic in 2020. COVID-19 is a new betacoronavirus and a variant of severe acute respiratory syndrome coronavirus 2 (SARA- CoV-2). Rapid, accurate and reliable diagnosis of COVID-19 will prevent the spread and allow for appropriate management. The main objective of this systematic review is to identify, appraise and summarise the published evidence on the diagnostic performance and effectiveness of SARS-CoV-2 virus in the diagnosis of current or previous COVID-19 using real-time polymerase chain reaction (RT-PCR) assay in low-and middle-income countries (LMICs). Methods: We will search MEDLINE/PubMed, EMBASE, BIOSIS, LILACS, Cochrane Infectious Diseases Group Specialised Register (CIDG SR), Global Health, and CINAHL for published studies for the diagnosis of COVID-19 using real-time polymerase chain reaction assay in LMICs There will be no restriction regarding the language, date of publication, and publication status. We will include retrospective, cross-sectional and cohort observational studies will be included in the review. Selection of studies, data extraction and management, assessment of risk of bias, and quality of evidence will be performed by two independent reviewers (EB and BC). A third researcher (GM) will be consulted in case of discrepancies. Depending on the availability and quality of the data, a meta-analysis will be performed. Otherwise, findings will be qualitatively reported. Discussion: To our knowledge, this is the first systematic review and meta-analysis to assess the uptake of RT-PCR assay for SARS-CoV-2 detection from clinical samples in human in LMICs. This review will make available evidence on the uptake, accuracy, approach, and interpretation of results of this assay in the context of COVID-19 diagnosis which will meet an urgent need, considering the diagnostic challenges of RT-PCR assay for COVID-19 diagnosis in humans. Systematic review registration: PROSPERO CRD42021271894

scholarly journals Development of a Conventional RT-PCR Assay for Rapid Detection of Porcine Deltacoronavirus with the Same Detection Limit as a SYBR Green-Based Real-Time RT-PCR Assay

2018 ◽  
Vol 2018 ◽  
pp. 1-7
Author(s):  
Lei Ma ◽  
Fanwen Zeng ◽  
Bihong Huang ◽  
Feng Cong ◽  
Ren Huang ◽  
...  
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Limit Of Detection ◽  
Sybr Green ◽  
Watery Diarrhea ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Rna Molecules ◽  
Positive Rate

Porcine deltacoronavirus (PDCoV) is a newly discovered coronavirus, which belongs to the family Coronaviridae. It causes watery diarrhea, vomiting, and dehydration in newborn piglets. A sensitive RT-PCR method is urgently required to detect PDCoV infection. In this study, we developed and evaluated a conventional RT-PCR assay and a SYBR green-based real-time RT-PCR assay that targeted the PDCoV n gene. Both assays are specific and have the same limit of detection at 2 × 101 copies of RNA molecules per reaction. Eighty-four clinical samples were subjected to both conventional RT-PCR and real-time RT-PCR, and the same positive rate (41.7%) was achieved, which was much higher than the positive rate (26.2%) using a previously described one-step RT-PCR technique. In summary, a conventional RT-PCR technique was successfully established for the detection of PDCoV with the same detection limit as a SYBR green-based real-time RT-PCR assay.

scholarly journals Establishment of a Real-time RT-PCR Assay for Avian Encephalomyelitis Virus

2016 ◽  
Vol 72 (7) ◽  
pp. 418-422
Author(s):  
Qinghong Xue ◽  
Hui Guo ◽  
Zhongze Feng ◽  
Jia Wang
Keyword(s):  
Real Time ◽  
Disease Virus ◽  
Rapid Detection ◽  
Prevention And Control ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Agar Diffusion Test ◽  
Encephalomyelitis Virus ◽  
And Control

Avian encephalomyelitis is an epidemic disease caused by avian encephalomyelitis virus (AEV), which exerts egg drop, slow growth, decreased egg hatchability, rapid tremors and ataxia of the head and neck in chicks and laying hens. The availability of a robust diagnostic assay to confirm the infection of AEV is important for its prevention and control. Thus, this study aimed to develop and validate a rapid Real-time RT-PCR assay for the detection and quantification of AEV. The authors developed and validated a TaqMan-based fluorescent real-time RT-PCR with sensitivity, specificity and reproducibility for rapid detection of EAV. This assay was then applied on 52 clinical samples from 37 suspected chickens. The minimal detection limit of this method was 20 copies. No cross-reactivity with Newcastle disease virus (NDV), avian influenza virus (AIV), Marek’s disease virus (MDV) or avian leukosis virus (ALV) was observed. The intra- and inter-assay coefficients of variation were both below 2%. Of the 52 clinical samples analyzed using this assay, 33 samples were positive for AEV, yielding an agreement rate of 91.2% with RT-PCR and 88.2% with the agar diffusion test. In conclusion, we have successfully developed and validated a sensitive Real-time RT-PCR for rapid detection of AEV, which could be used in the early diagnosis, prevention and control, and epidemiological investigation of AEV.

scholarly journals Application of a SYBR® Green One Step Real-time RT-PCR Assay to Detect Type 1 Porcine Reproductive and Respiratory Syndrome Virus

2014 ◽  
Vol 76 (10) ◽  
pp. 1411-1413 ◽  
Author(s):  
Hiroshi ISEKI ◽  
Michihiro TAKAGI ◽  
Yoshiko KURODA ◽  
Ken KATSUDA ◽  
Osamu MIKAMI ◽  
...  
Keyword(s):  
Real Time ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
One Step

scholarly journals Detection of SARS Coronavirus in Patients with Severe Acute Respiratory Syndrome by Conventional and Real-Time Quantitative Reverse Transcription-PCR Assays

2004 ◽  
Vol 50 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Leo L M Poon ◽  
Kwok Hung Chan ◽  
On Kei Wong ◽  
Timothy K W Cheung ◽  
Iris Ng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
N Gene ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Copy Numbers ◽  
Stool Samples ◽  
Pcr Assays

Abstract Background: A novel coronavirus (CoV) was recently identified as the agent for severe acute respiratory syndrome (SARS). We compared the abilities of conventional and real-time reverse transcription-PCR (RT-PCR) assays to detect SARS CoV in clinical specimens. Methods: RNA samples isolated from nasopharyngeal aspirate (NPA; n = 170) and stool (n = 44) were reverse-transcribed and tested by our in-house conventional RT-PCR assay. We selected 98 NPA and 37 stool samples collected at different times after the onset of disease and tested them in a real-time quantitative RT-PCR specific for the open reading frame (ORF) 1b region of SARS CoV. Detection rates for the conventional and real-time quantitative RT-PCR assays were compared. To investigate the nature of viral RNA molecules in these clinical samples, we determined copy numbers of ORF 1b and nucleocapsid (N) gene sequences of SARS CoV. Results: The quantitative real-time RT-PCR assay was more sensitive than the conventional RT-PCR assay for detecting SARS CoV in samples collected early in the course of the disease. Real-time assays targeted at the ORF 1b region and the N gene revealed that copy numbers of ORF 1b and N gene sequences in clinical samples were similar. Conclusions: NPA and stool samples can be used for early diagnosis of SARS. The real-time quantitative RT-PCR assay for SARS CoV is potentially useful for early detection of SARS CoV. Our results suggest that genomic RNA is the predominant viral RNA species in clinical samples.

scholarly journals Development of TaqMan-based real-time RT-PCR assay based on N gene for the quantitative detection of feline morbillivirus

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Siti Tasnim Makhtar ◽  
Sheau Wei Tan ◽  
Nur Amalina Nasruddin ◽  
Nor Azlina Abdul Aziz ◽  
Abdul Rahman Omar ◽  
...  
Keyword(s):  
Real Time ◽  
Measles Virus ◽  
Clinical Sample ◽  
N Gene ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
High Morbidity ◽  
Canine Distemper ◽  
Rt Pcr ◽  
Pcr Assay

Abstract Background Morbilliviruses are categorized under the family of Paramyxoviridae and have been associated with severe diseases, such as Peste des petits ruminants, canine distemper and measles with evidence of high morbidity and/or could cause major economic loss in production of livestock animals, such as goats and sheep. Feline morbillivirus (FeMV) is one of the members of Morbilliviruses that has been speculated to cause chronic kidney disease in cats even though a definite relationship is still unclear. To date, FeMV has been detected in several continents, such as Asia (Japan, China, Thailand, Malaysia), Europe (Italy, German, Turkey), Africa (South Africa), and South and North America (Brazil, Unites States). This study aims to develop a TaqMan real-time RT-PCR (qRT-PCR) assay targeting the N gene of FeMV in clinical samples to detect early phase of FeMV infection. Results A specific assay was developed, since no amplification was observed in viral strains from the same family of Paramyxoviridae, such as canine distemper virus (CDV), Newcastle disease virus (NDV), and measles virus (MeV), and other feline viruses, such as feline coronavirus (FCoV) and feline leukemia virus (FeLV). The lower detection limit of the assay was 1.74 × 104 copies/μL with Cq value of 34.32 ± 0.5 based on the cRNA copy number. The coefficient of variations (CV) values calculated for both intra- and inter-assay were low, ranging from 0.34–0.53% and 1.38–2.03%, respectively. In addition, the clinical sample evaluation using this assay showed a higher detection rate, with 25 (35.2%) clinical samples being FeMV-positive compared to 11 (15.5%) using conventional RT-PCR, proving a more sensitive assay compared to the conventional RT-PCR. Conclusions The TaqMan-based real-time RT-PCR assay targeting the N gene described in this study is more sensitive, specific, rapid, and reproducible compared to the conventional RT-PCR assay targeting the N gene, which could be used to detect early infection in cats.

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