scholarly journals Comparison of bluetongue virus detection and quantitation methods in south India

2014 ◽  
Vol 8 (10) ◽  
pp. 1307-1312
Author(s):  
Subhra Subhadra ◽  
Subrat Kumar ◽  
Veluvarthy VS Suryanarayana ◽  
Daggupati Sreenivasulu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Disease Diagnosis ◽  
Sybr Green ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Animal Blood ◽  
Specificity And Sensitivity

Introduction: Bluetongue (BT), a vector-borne viral disease, primarily affects sheep. Of the 26 serotypes of BTV identified so far, 22 are reported to be circulating in India. Due to an increase in vector population and delays in disease diagnosis, the BT control program heavily relies on rapid and confirmatory diagnosis. Polymerase chain reaction (PCR)-based real-time detection assays may be an ideal method to detect the BTV genome in animal blood at an early stage of infection. Methodology: In this study, a SYBR green-based real-time RT-PCR assay was evaluated, validated, and compared with conventional RT-PCR. The specificity and sensitivity of an assay using BTV-2 RNA extracted from tenfold serially diluted (starting from 1.0 TCID50/mL) cell culture virus was also evaluated. Results: While conventional RT-PCR could detect 3.16×102 TCID50 of virus/mL, the real-time PCR test had a detection limit of 3.16×10-4 TCID50/mL. Melting curve analysis indicated the absence of non-specific amplification (R2 = 0.987). Out of the 32 infected blood samples examined, 24 tested positive for BTV RNA. Seven that were found negative through conventional PCR tested positive through real-time PCR. Conclusions: These results showed that the SYBR green-based real-time PCR assay is rapid, sensitive, and equally specific in the diagnosis of BT in BTV-affected animals.

Duplex Real-time PCR assay and SYBR green I melting curve analysis for molecular identification of HPV genotypes 16, 18, 31, 35, 51 and 66

2015 ◽  
Vol 29 (1) ◽  
pp. 13-18 ◽  
Author(s):  
D. Tsakogiannis ◽  
M. Papacharalampous ◽  
E. Toska ◽  
Z. Kyriakopoulou ◽  
T.G. Dimitriou ◽  
...  
Keyword(s):  
Real Time ◽  
Molecular Identification ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Curve Analysis ◽  
Sybr Green ◽  
Sybr Green I ◽  
Melting Curve Analysis ◽  
Pcr Assay ◽  
Hpv Genotypes

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

2018 ◽  
Vol 56 (8) ◽  
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.

A novel duplex SYBR Green real-time PCR with melting curve analysis method for beef adulteration detection

2021 ◽  
Vol 338 ◽  
pp. 127932
Author(s):  
Jiapeng Li ◽  
Yixuan Wei ◽  
Jinchun Li ◽  
Ruixi Liu ◽  
Suigen Xu ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Curve Analysis ◽  
Sybr Green ◽  
Melting Curve Analysis ◽  
Analysis Method

scholarly journals Rapid diagnosis of human brucellosis by SYBR Green I-based real-time PCR assay and melting curve analysis in serum samples

2005 ◽  
Vol 11 (9) ◽  
pp. 713-718 ◽  
Author(s):  
M.I. Queipo-Ortuño ◽  
J.D. Colmenero ◽  
J.M. Reguera ◽  
M.A. García-Ordoñez ◽  
M.E. Pachón ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Rapid Diagnosis ◽  
Curve Analysis ◽  
Sybr Green I ◽  
Melting Curve Analysis ◽  
Human Brucellosis ◽  
Pcr Assay ◽  
Serum Samples

scholarly journals MULTIPLEX SYBR GREEN ASSAY FOR CORONAVIRUS DETECTION USING FAST REAL-TIME RT-PCR

2020 ◽  
Vol 51 (2) ◽  
pp. 556-564
Author(s):  
Salah & et al.
Keyword(s):  
Respiratory Tract ◽  
Real Time ◽  
Melting Curve ◽  
First Record ◽  
Sybr Green ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Efficient Detection ◽  
Upper Respiratory ◽  
Tract Infections

This study was aimed to provide a local database for detection of coronavirus (CoV) species in suspect individual with respiratory tract infections like influenza type A and a tuberculosis using multiplex Sybr green reverse transcriptase real-time PCR (rRT-PCR) technique. A total of 500 samples was collected from individuals suffering from upper and/or lower respiratory tract diseases for testing of 4 CoV species (229E, OC43, NL63, and HKU1). RNA extracted, amplified and subsequent the positive samples sequencing. The results showed melting curve analysis (Tm) of the specific amplicons (79.73±0.36) and 9% positive for CoVs  and some of them have other co-infection such as influenza virus 26.67%, and TB 11.11%. On the other hands, the CoVs were detected 4.62% in upper respiratory samples and 20.39% with lower respiratory samples. Sequencing results pointed out two isolates were CoV-NL63 and four isolates were CoV-229E, with first record accession number MN086823.1 and MN086824.1, respectively in GenBank. In conclusion, this rRT-PCR showed the rapid and efficient detection of CoVs with few copies number. This allows being used for the diagnosis of CoVs along with other respiratory viruses in a multiplex assay to reduce processing time. Subsequent applied nested RT-PCR to overcome the low viral load.

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