Fast and Type-Specific Analysis of Herpes Simplex Virus Types 1 and 2 by Rapid PCR and Fluorescence Melting-Curve-Analysis

Infection ◽  
2000 ◽  
Vol 28 (2) ◽  
pp. 85-91 ◽  
Author(s):  
G. Schalasta ◽  
A. Arents ◽  
M. Schmid ◽  
R.W. Braun ◽  
G. Enders
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Rapid Pcr ◽  
Specific Analysis ◽  
Fluorescence Melting Curve Analysis ◽  
Simplex Virus

scholarly journals Diagnosis of Herpes Simplex Virus Infections in the Clinical Laboratory by LightCycler PCR

2000 ◽  
Vol 38 (2) ◽  
pp. 795-799 ◽  
Author(s):  
Mark J. Espy ◽  
James R. Uhl ◽  
P. Shawn Mitchell ◽  
Jill N. Thorvilson ◽  
Kathleen A. Svien ◽  
...  
Keyword(s):  
Cell Culture ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Cultures ◽  
Melting Curve ◽  
Clinical Manifestations ◽  
Melting Curve Analysis ◽  
Shell Vial ◽  
Pcr Products ◽  
Simplex Virus

Herpes simplex virus (HSV) causes several clinical manifestations in both normal and immunocompromised hosts; this agent is the most frequently detected virus in diagnostic laboratories. Recovery of the virus in cell culture is considered the “gold standard” for detection of this virus from sources other than cerebrospinal fluid. LightCycler is a newly developed, commercially available system designed to rapidly perform PCR, with real-time detection of PCR products by a fluorescence resonance energy transfer assay. We compared the detection of HSV for 200 specimens (number of genital specimens, 160; number of dermal specimens, 38; number of ocular specimens, 2) by shell vial cell cultures (MRC-5) and by LightCycler PCR. Of a total of 88 (44%) HSV strains detected, 69 (78%) were detected by both shell vial cell cultures and LightCycler PCR (DNA polymerase target). A total of 19 (22%) specimens were detected exclusively by LightCycler PCR. No specimens were positive by the shell vial assay only. All 19 discrepant samples had HSV DNA detected by an independent PCR directed to the thymidine kinase gene of the virus. The melting curve analysis feature of the LightCycler instrument identified identical genotype results for HSV type 1 (HSV-1) and HSV-2 from 84 of 88 (96%) positive samples. Specimens can be extracted, target HSV DNA can be amplified, and HSV PCR products can be identified by genotype within 2 h after receipt of specimen into the laboratory. The increased level of accurate identification (all 88 positive samples) compared with that of shell vial cell culture (69 of 88 samples identified as positive) and the agreement of LightCycler PCR results with all shell vial positive results indicate the potential for routine implementation of this technology for laboratory diagnosis of HSV infections.

Direct comparison study between droplet digital PCR and a combination of allele-specific PCR, asymmetric rapid PCR and melting curve analysis for the detection of BRAF V600E mutation in plasma from melanoma patients

2020 ◽  
Vol 58 (11) ◽  
pp. 1799-1807 ◽  
Author(s):  
Eleni Tzanikou ◽  
Verena Haselmann ◽  
Athina Markou ◽  
Angelika Duda ◽  
Jochen Utikal ◽  
...  
Keyword(s):  
Melting Curve ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Curve Analysis ◽  
Response To Therapy ◽  
Braf V600e ◽  
Melting Curve Analysis ◽  
Comparison Study ◽  
Rapid Pcr ◽  
Melanoma Patients

AbstractBackgroundIn metastatic melanoma, 40%–50% of patients harbor a BRAF V600E mutation and are thereby eligible to receive a combined BRAF/MEK inhibitor therapy. Compared to standard-of-care tissue-based genetic testing, analysis of circulating tumor DNA (ctDNA) from blood enables a comprehensive assessment of tumor mutational status in real-time and can be used for monitoring response to therapy. The aim of our study was to directly compare the performance of two highly sensitive methodologies, droplet digital PCR (ddPCR) and a combination of ARMS/asymmetric-rapid PCR/melting curve analysis, for the detection of BRAF V600E in plasma from melanoma patients.MethodsCell-free DNA (cfDNA) was isolated from 120 plasma samples of stage I to IV melanoma patients. Identical plasma-cfDNA samples were subjected to BRAF V600E mutational analysis using in parallel, ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis.ResultsBRAF V600E mutation was detected in 9/117 (7.7%) ctDNA samples by ddPCR and in 22/117 (18.8%) ctDNA samples by the combination of ARMS/asymmetric- rapid PCR/melting curve analysis. The concordance between these two methodologies was 85.5% (100/117). The comparison of plasma-ctDNA analysis using ddPCR and tissue testing revealed an overall agreement of 79.4% (27/34), while the corresponding agreement using the combination of ARMS/asymmetric-rapid PCR/melting curve analysis was 73.5% (25/34). Moreover, comparing the detection of BRAF-mutant ctDNA with the clinics, overall agreement of 87.2% (48/55) for ddPCR and 79.2% (42/53) was demonstrated. Remarkably, the duration of sample storage was negatively correlated with correctness of genotyping results highlighting the importance of pre-analytical factors.ConclusionsOur direct comparison study has shown a high level of concordance between ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis for the detection of BRAF V600E mutations in plasma.

Probe-Based Fluorescence Melting Curve Analysis for Differentiating Larimichthys polyactis and Larimichthys crocea

2016 ◽  
Vol 9 (7) ◽  
pp. 2036-2041 ◽  
Author(s):  
Jeong Jin Ahn ◽  
Youngjoo Kim ◽  
Ji Young Hong ◽  
Gi Won Kim ◽  
Seol Young Kim ◽  
...  
Keyword(s):  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Larimichthys Crocea ◽  
Larimichthys Polyactis ◽  
Fluorescence Melting Curve Analysis

scholarly journals Detection of Herpes Simplex and Varicella-Zoster Virus in Clinical Specimens by Multiplex Real-Time PCR and Melting Curve Analysis

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Yun Ji Hong ◽  
Mi Suk Lim ◽  
Sang Mee Hwang ◽  
Taek Soo Kim ◽  
Kyoung Un Park ◽  
...  
Keyword(s):  
Herpes Simplex ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Laboratory ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Clinical Specimens ◽  
Varicella Zoster ◽  
Hsv 1

Herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2), and varicella-zoster virus (VZV) are common agents resulting in various forms of clinical manifestation from skin vesicle to disseminated viral infection. The aim of the present study was to develop a real-time PCR and melting curve analysis which detect and differentiate HSV-1, HSV-2, and VZV, to compare with PCR-RFLP using clinical specimens, and to introduce the 4-year experience in the clinical laboratory. Three pairs of primers for HSV-1, HSV-2, and VZV were designed. Primers for human endogenous retrovirus-3 (HERV-3), an internal control, were adopted. A hundred selected specimens and many clinical specimens were tested for methods comparison and assay validation. Increased sensitivity and specificity were obtained from real-time PCR. In review of results of clinical specimens submitted to clinical laboratory, a total of 46 of 3,513 specimens were positive in cerebrospinal fluids, blood, skin vesicles, genital swabs, aqueous humor, and ear discharge. Thus, this method could be a rapid and accurate alternative to virus culture and other molecular tests for detection and typing of HSV-1, HSV-2, and VZV.

scholarly journals Detection of Eight Respiratory Bacterial Pathogens Based on Multiplex Real-Time PCR with Fluorescence Melting Curve Analysis

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Liuyang Hu ◽  
Bing Han ◽  
Qin Tong ◽  
Hui xiao ◽  
Donglin Cao
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Bacterial Pathogens ◽  
Melting Curve ◽  
Cost Effective ◽  
Culture Method ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Fluorescence Melting Curve Analysis

Background and Objective. Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis are primary respiratory bacterial pathogens contributing to morbidity and mortality in developing countries. This study evaluated the diagnostic performance of multiplex real-time PCR with fluorescence melting curve analysis (MCA) assay, which was used to detect eight respiratory bacterial pathogens simultaneously. Methods. A total of 157 sputum specimens were examined by multiplex real-time with fluorescence MCA, and the results were compared with the conventional culture method. Results. Multiplex real-time PCR with fluorescence MCA specifically detected and differentiated eight respiratory bacterial pathogens by different melting curve peaks for each amplification product within 2 hours and exhibited high repeatability. The limit of detection ranged from 64 to 102 CFU/mL in the multiplex PCR system. Multiplex real-time PCR with fluorescence MCA showed a sensitivity greater than 80% and a 100% specificity for each pathogen. The kappa correlation of eight bacteria ranged from 0.89 to 1.00, and the coefficient of variation ranged from 0.05% to 0.80%. Conclusions. Multiplex real-time PCR with fluorescence MCA assay is a sensitive, specific, high-throughput, and cost-effective method to detect multiple bacterial pathogens simultaneously.

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