Simultaneous detection and genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCR and amplicon melting curve analysis using SYBR Green

2008 ◽  
Vol 85 (1) ◽  
pp. 184-193 ◽  
Author(s):  
E. Martínez ◽  
P. Riera ◽  
M. Sitjà ◽  
Y. Fang ◽  
S. Oliveira ◽  
...  
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
Melting Curve ◽  
Curve Analysis ◽  
Sybr Green ◽  
Respiratory Syndrome Virus ◽  
Melting Curve Analysis ◽  
Rt Pcr

Detection of SYT-SSX Rearrangements in Synovial Sarcomas by Real-Time One-Step RT-PCR

2005 ◽  
Vol 8 (2) ◽  
pp. 162-167 ◽  
Author(s):  
Marina N. Nikiforova ◽  
Pamela Groen ◽  
George Mutema ◽  
Yuri E. Nikiforov ◽  
David Witte
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
Melting Curve ◽  
Soft Tissue Sarcomas ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Simple Method ◽  
Synovial Sarcomas ◽  
One Step

Synovial sarcomas are aggressive tumors of adolescent and young adults that account for up to 10% of soft tissue sarcomas. Cytogenetically, they are characterized by translocation t(X;18), which is found in more than 95% of tumors. In most cases, it results in fusion of the SYT gene with the SSX1 or SSX2 gene, thus creating SYT-SSX1 or SYT-SSX2 rearrangement. The 2 types of gene fusion have been correlated with histologic variants and prognosis of synovial sarcomas. In this study, we developed a simple and rapid method for the simultaneous detection of SYT-SSX1 and SYT-SSX2 rearrangements by using a LightCycler real-time one-step reverse transcriptase polymerase chain reaction (RT-PCR) technology (Roche). Oligonucleotide probes were designed so that the donor probe would span a fusion point and the acceptor probe would be complementary to the SSX1 sequence but have 2 nucleotide mismatches with SSX2 sequence. Such a design allows simultaneous amplification of 2 types of rearrangement in the same reaction but distinguishes them based on differences in melting temperature detected by melting curve analysis after PCR. With this method, 27 tumors (9 synovial sarcomas and 18 nonsynovial sarcomas) were studied and showed SYT-SSX1 rearrangement in 6 cases and SYT-SSX2 in 3 cases. These results had complete correlation with the finding of conventional RT-PCR and direct sequencing. In conclusion, we have developed a fast, accurate, and simple method for the detection of 2 major types of SYT-SSX rearrangement by using LightCycler RT-PCR and melting curve analysis.

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 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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