scholarly journals Development of a nanoparticle-assisted PCR assay to distinguish canine coronaviruses I and II

2020 ◽  
Vol 33 (1) ◽  
pp. 104-107
Author(s):  
Tong Qin ◽  
Jing Wang ◽  
Shang-Jin Cui
Keyword(s):  
Mixed Infection ◽  
Annealing Temperature ◽  
Clinical Samples ◽  
Rt Pcr ◽  
M Gene ◽  
Specific Detection ◽  
Pcr Assay ◽  
Reaction Conditions ◽  
Novel Method ◽  
Beijing China

Nanoparticle-assisted PCR (nanoPCR) is a novel method for the simple, rapid, and specific detection of viruses. We developed a nanoPCR method to detect and differentiate canine coronavirus I (CCoV I) and II (CCoV II). Primer pairs were designed against the M gene conserved region of CCoV I and CCoV II, producing specific fragments of 239 bp (CCoV I) and 105 bp (CCoV II). We optimized the annealing temperature and primer concentrations for the CCoV nanoPCR assay and assessed its sensitivity and specificity. Under optimized nanoPCR reaction conditions, the detection limits were 6.47 × 101 copies/μL for CCoV I and 6.91 × 102 copies/μL for CCoV II. No fragments were amplified using other canine viruses as templates. The sensitivity of the nanoPCR assay was 100-fold higher than that of a conventional RT-PCR assay. Among 60 clinical samples collected from Beijing, China, the assay detected 12% positive for CCoV I and 48% positive for CCoV II. Our nanoPCR method is an effective method to rapidly detect CCoV I and CCoV II alone, or as a mixed infection, in dogs.

scholarly journals The application of RT-PCR assay for the detection of Apple stem pitting virus and Apple stem grooving virus in four apple cultivars

2012 ◽  
Vol 38 (No. 1) ◽  
pp. 13-17 ◽  
Author(s):  
J.K. Kundu
Keyword(s):  
Mixed Infection ◽  
Rt Pcr ◽  
Pome Fruit ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Apple Stem Grooving Virus ◽  
Apple Stem ◽  
Apple Cultivars ◽  
Apple Stem Pitting Virus ◽  
Stem Pitting

The reverse transcription polymerace chain reaction (RT-PCR) assay was successfully used for the detection of Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) in four apple cultivars of a 25 years old orchard. These two main pome fruit viruses were detected frequently in all tested apple cultivars. ASGV and ASPV occurred in as many as 16 trees (in the cultivar Spartan) and 13 trees (in the cultivar Idared) out of 20 tested trees, respectively. Mixed infection by ASGV and ASPV was found in all tested cultivars (as many as 9 out of 20 tested trees of the cultivar Spartan).

Development of a novel SYBR green I-based quantitative RT-PCR assay for Senecavirus A detection in clinical samples of pigs

2020 ◽  
Vol 53 ◽  
pp. 101643
Author(s):  
Suyu Mu ◽  
Sahibzada Waheed Abdullah ◽  
Yun Zhang ◽  
Shichong Han ◽  
Huichen Guo ◽  
...  
Keyword(s):  
Sybr Green ◽  
Sybr Green I ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay

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 Improved Detection of Rhinoviruses in Clinical Samples by Using a Newly Developed Nested Reverse Transcription-PCR Assay

1999 ◽  
Vol 37 (3) ◽  
pp. 524-530 ◽  
Author(s):  
Arno C. Andeweg ◽  
Theo M. Bestebroer ◽  
Martijn Huybreghs ◽  
Tjeerd G. Kimman ◽  
Jan C. de Jong
Keyword(s):  
Reverse Transcription ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Noncoding Regions ◽  
Reverse Transcription Pcr ◽  
Highly Sensitive ◽  
Virus Culture ◽  
Culture Positive

This paper describes the development and evaluation of a new nested reverse transcription (RT)-PCR for the detection of rhinovirus in clinical samples. The nucleotide sequences of the 5′ noncoding regions of 39 rhinoviruses were determined in order to map the most conserved subregions. We designed a set of rhinovirus-specific primers and probes directed to these subregions and developed a new nested RT-PCR. The new assay includes an optimal RNA extraction method and amplicon identification with probe hybridization to discriminate between rhinoviruses and the closely related enteroviruses. It proved to be highly sensitive and specific. When tested on a dilution series of cultured viruses, the new PCR protocol scored positive at 10- to 100-fold-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory disease patients who had consulted their general practitioners, the new assay demonstrated a rhinovirus in 24% of the specimens, including all culture-positive samples, whereas the previously used PCR assay or virus culture detected a rhinovirus in only 3.5 to 6% of the samples. This new assay should help determine the disease burden associated with rhinovirus infections.

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 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 Rapid Detection of 2019 Novel Coronavirus SARS-CoV-2 Using a CRISPR-based DETECTR Lateral Flow Assay

2020 ◽  
Author(s):  
James P. Broughton ◽  
Xianding Deng ◽  
Guixia Yu ◽  
Clare L. Fasching ◽  
Jasmeet Singh ◽  
...  
Keyword(s):  
Low Cost ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
The Us ◽  
Comparable Performance ◽  
Novel Coronavirus ◽  
Reference Samples

ABSTRACTAn outbreak of novel betacoronavirus, SARS-CoV-2 (formerly named 2019-nCoV), began in Wuhan, China in December 2019 and the COVID-19 disease associated with infection has since spread rapidly to multiple countries. Here we report the development of SARS-CoV-2 DETECTR, a rapid (∼30 min), low-cost, and accurate CRISPR-Cas12 based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated this method using contrived reference samples and clinical samples from infected US patients and demonstrated comparable performance to the US CDC SARS-CoV-2 real-time RT-PCR assay.

DETECTION OF RESPIRATORY SYNCITIAL VIRUS (RSV) IN LOWER ACUTE RESPIRATORY INFECTIONS BY NESTED REVERSE TRANSCRIPTION- POLYMERASE CHAIN REACTION

2011 ◽  
pp. 11-15
Author(s):  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Respiratory Infections ◽  
Clinical Samples ◽  
Acute Respiratory Infections ◽  
Rt Pcr ◽  
Minimal Level ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

Objective: To develop and apply a nested reverse transcription- polymerase chain reaction (nested RT-PCR) for detection of RSV in lower acute respiratory infections. Materials and methods: A nested reverse transcription- polymerase chain reaction was used to amplify a sequence of the F gene in the RSV genomic RNA, optimized and compared the sensitivity and specificity of this assay with the control samples and then applied this procedure for diagnosing RSV from clinical samples. Results: This nested RT-PCR assay amplified the specific target fragment of RSV RNA and did not amplify any sequence of genomes of the tested common viruses and bacteria causing respiratory infections. The minimal level of detection of this procedure was 102 copies/ml. Results for detection of RSV on 109 samples of throat swabs or nasopharyngeal swabs from children with lower respiratory infections showed that twenty seven patients were positive with RSV ( 24.8%), among which six out of 30 (20%) were with bronchitis, seven out of 26 ( 27%) were with bronchiolitis and fourteen out of 53 (26.4%) were with pneumonia. Conclusion: This nested RT-PCR was found to be useful and reliable for detection of RSV in respiratory infections.

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.

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