scholarly journals Development and validation of cost-effective one-step multiplex RT-PCR assay for detecting the SARS-CoV-2 infection using SYBR Green melting curve analysis

2021 ◽  
Author(s):  
Shovon Lal Sarkar ◽  
A. S. M. Rubayet Ul Alam ◽  
Prosanto Kumar Das ◽  
Md. Hasan Ali Pramanik ◽  
Hassan M. Al-Emran ◽  
...  
Keyword(s):  
Internal Control ◽  
Melting Curve ◽  
Cost Effective ◽  
Curve Analysis ◽  
Sybr Green ◽  
Taqman Probe ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
One Step ◽  
Commercial Kit

TaqMan probe-based expensive commercial real-time (RT) PCR kits are being used in COVID-19 diagnosis. The unprecedented scale of SARS-CoV-2 infections has urgently needed to meet the challenge of testing more persons at a reasonable cost. This study developed a rapid, simple, and cost-effective alternative diagnostic method based on melting curve analysis of SYBR green multiplex assay with a host-specific internal control. A total of 90 randomly selected samples were used for comparing the assay with an available commercial kit to analyse the variation and validity of this in-house developed method. Our customized designed primers specifically detected the virus as similar to commercial kit manufactured by Sansure Biotech Inc. We optimized separately the N, E, S, and RdRp genes by SYBR Green RT-PCR method based on melting curve analysis. Afterwards, a multiplex COVID-19 diagnosis method targeting N and E genes of the virus along with the β-actin gene of the host as an internal control has been established. The total run-time of our proposed method was less than 90 minutes. The cost of each sample processing was less than $2. Overall, this one-step and one-tube method can revolutionize the COVID-19 diagnosis in developing countries.

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.

scholarly journals A duplex real-time PCR for the detection of Streptococcus pneumoniae and Neisseria meningitidis in cerebrospinal fluid

2016 ◽  
Vol 10 (01) ◽  
pp. 53-61 ◽  
Author(s):  
Idrissa Diawara ◽  
Khalid Katfy ◽  
Khalid Zerouali ◽  
Houria Belabbes ◽  
Naima Elmdaghri
Keyword(s):  
Cerebrospinal Fluid ◽  
Bacterial Meningitis ◽  
Patient Management ◽  
Melting Curve ◽  
Curve Analysis ◽  
Sybr Green ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Pcr Method

Introduction: Acute bacterial meningitis is one of the most severe infectious diseases. Rapid, accurate, and inexpensive diagnosis of bacterial meningitis is crucial for patient management. This study describes a duplex real-time (RT) PCR assay for detection of Neisseria meningitidis and Streptococcus pneumoniae in the cerebrospinal fluid (CSF) for meningitis diagnosis using SYBR Green-based RT-PCR method coupled with melting curve analysis. Methodology: We used SYBR Green-based RT-PCR method coupled with melting curve analysis to detect S. pneumoniae and N. meningitidis in CSF samples. The sensitivity, specificity, and limit of detection were determined. The gold standard for routine tests of CSF analysis is direct examination, culture, and/or latex agglutination. The assay was evaluated on 132 CSF samples to measure clinical sensitivity. Results: A duplex RT-PCR assay for N. meningitidis and S. pneumoniae detection in CSF was evaluated. Two peaks at different melting temperatures (87.5°C and 85.5°C) for N. meningitidis and S. pneumoniae, respectively, were obtained. The sensitivity of RT-PCR was 100% (95% confidence limits [CI] = 82.4–100) for N. meningitidis and 100% (95% CI = 85.1–100) for S. pneumoniae. Specificity was the same (100%) for the bacteria (95% CI = 88.6–100). The percentage of cases accurately diagnosed with meningitis caused by N. meningitidis and S. pneumoniae increased to 50.7% and 28.6%, respectively, when RT-PCR was added to the standard microbiologic methods. Conclusions: Duplex RT-PCR and melting curve analysis with SYBR Green is an inexpensive, sensitive, and specific method to rapidly diagnose bacterial meningitis. Accurate identification of the bacterial causative agents will improve patient management and epidemiological investigations.

scholarly journals Cost-effective and fast KIR gene-content genotyping by multiplex melting curve analysis

HLA ◽  
2018 ◽  
Vol 92 (6) ◽  
pp. 384-391
Author(s):  
Leonardo M. Amorim ◽  
Tiago H. S. Santos ◽  
Jill A. Hollenbach ◽  
Paul J. Norman ◽  
Wesley M. Marin ◽  
...  
Keyword(s):  
Melting Curve ◽  
Cost Effective ◽  
Curve Analysis ◽  
Gene Content ◽  
Melting Curve Analysis ◽  
Kir Gene

scholarly journals Rapid base-specific calling of SARS-CoV-2 variants of concern using combined RT-PCR melting curve screening and SIRPH technology

2021 ◽  
Author(s):  
Sascha Tierling ◽  
Kathrin Kattler ◽  
Markus Vogelgesang ◽  
Thorsten Pfuhl ◽  
Stefan Lohse ◽  
...  
Keyword(s):  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Single Nucleotide ◽  
Specific Identification ◽  
Rp Hplc ◽  
Novel Variants ◽  
Reliable Detection ◽  
Cost Efficient

The emergence of novel variants of concern of SARS-CoV-2 demands a fast and reliable detection of such variants in local populations. Here we present a cost-efficient and fast workflow combining a pre-screening of SARS-CoV-2 positive samples using RT-PCR melting curve analysis with multiplexed IP-RP-HPLC-based single nucleotide primer extensions (SIRPH). The entire workflow from positive SARS-CoV-2 testing to base-specific identification of variants requires about 24 h. We applied the sensitive method to monitor the local VOC outbreaks in a few hundred positive samples collected in a confined region of Germany.

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