Dataset Information

Sequencing and resolution of amplified herpes simplex virus DNA with intermediate melting curves as genotype 1 or 2 by LightCycler PCR assay.

ABSTRACT: DNA from 101 specimens containing herpes simplex virus (HSV) produced atypical intermediate melting curves compared with those expected for HSV type 1 or HSV type 2 subsequent to real-time PCR. Nucleic acid sequence analysis of amplified target DNA revealed 1- or 3-bp polymorphisms in the probe region which allowed designation of these viruses as HSV genotype 1 or HSV genotype 2. These two subpopulations of HSV were also identified as HSV genotype 1 or HSV genotype 2 using another commercially available PCR method. Amplified HSV target DNA producing intermediate melting curves could be designated as HSV genotype 1 or HSV genotype 2 without performing sequencing or another PCR method with 96/101 (95%) specimens by adding known intermediate HSV DNA characteristic for the two subpopulations as controls.

SUBMITTER: Issa NC

PROVIDER: S-EPMC1081363 | biostudies-literature | 2005 Apr

REPOSITORIES: biostudies-literature

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Sequencing and resolution of amplified herpes simplex virus DNA with intermediate melting curves as genotype 1 or 2 by LightCycler PCR assay.

Issa Nicolas C NC Espy Mark J MJ Uhl James R JR Smith Thomas F TF

Journal of clinical microbiology 20050401 4

DNA from 101 specimens containing herpes simplex virus (HSV) produced atypical intermediate melting curves compared with those expected for HSV type 1 or HSV type 2 subsequent to real-time PCR. Nucleic acid sequence analysis of amplified target DNA revealed 1- or 3-bp polymorphisms in the probe region which allowed designation of these viruses as HSV genotype 1 or HSV genotype 2. These two subpopulations of HSV were also identified as HSV genotype 1 or HSV genotype 2 using another commercially a ...[more]

PMID: 15815006

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Project description:BACKGROUND: DNA melting curve analysis using double-stranded DNA-specific dyes such as SYTO9 produce complex and reproducible melting profiles, resulting in the detection of multiple melting peaks from a single amplicon and allowing the discrimination of different species. We compare the melting curves of several Naegleria and Cryptosporidium amplicons generated in vitro with in silico DNA melting simulations using the programs POLAND and MELTSIM., then test the utility of these programs for assay design using a genetic marker for toxin production in cyanobacteria. RESULTS: The SYTO9 melting curve profiles of three species of Naegleria and two species of Cryptosporidium were similar to POLAND and MELTSIM melting simulations, excepting some differences in the relative peak heights and the absolute melting temperatures of these peaks. MELTSIM and POLAND were used to screen sequences from a putative toxin gene in two different species of cyanobacteria and identify regions exhibiting diagnostic melting profiles. For one of these diagnostic regions the POLAND and MELTSIM melting simulations were observed to be different, with POLAND more accurately predicting the melting curve generated in vitro. Upon further investigation of this region with MELTSIM, inconsistencies between the melting simulation for forward and reverse complement sequences were observed. The assay was used to accurately type twenty seven cyanobacterial DNA extracts in vitro. CONCLUSION: Whilst neither POLAND nor MELTSIM simulation programs were capable of exactly predicting DNA dissociation in the presence of an intercalating dye, the programs were successfully used as tools to identify regions where melting curve differences could be exploited for diagnostic melting curve assay design. Refinements in the simulation parameters would be required to account for the effect of the intercalating dye and salt concentrations used in real-time PCR. The agreement between the melting curve simulations for different species of Naegleria and Cryptosporidium and the complex melting profiles generated in vitro using SYTO9 verified that the complex melting profile of PCR amplicons was solely the result of DNA dissociation. Other data outputs from these simulations were also used to identify the melting domains that contributed to the observed melting peaks for each of the different PCR amplicons.

Project description:BACKGROUND: The speed and sensitivity of real-time polymerase chain reaction (PCR) have made it a popular method for the detection of microbiological agents in both research and clinical specimens. For the detection and genotyping of herpes simplex virus (HSV) in clinical specimens, real-time PCR has proven to be faster, more sensitive and safer than earlier methods which included isolation of the virus in cell culture followed by immunofluorescence microscopy. While PCR-based assays for HSV detection posses clear advantages over these earlier techniques, certain aspects of the PCR method remain onerous. The process of extraction and purification of nucleic acid from clinical specimens prior to PCR is particularly cumbersome. Nucleic acid extraction is expensive, time-consuming and provides a step whereby specimens can become contaminated prior to their analysis. Herein, we investigate the necessity of nucleic acid extraction from swab-based clinical specimens for HSV detection by real-time PCR. We find that nucleic acid extraction is unnecessary for specific and sensitive detection of HSV in clinical specimens using real-time PCR. METHODS: Prospective (n = 36) and retrospective (n = 21) clinical specimens from various anatomical sites were analyzed for the presence of herpes simplex virus 1 or 2 by real-time PCR using the RealArt HSV 1/2 LC PCR Kit. Specimens were analyzed by PCR both before and following automated nucleic acid extraction. PCR using extracted and unextracted specimens was also compared to cell culture as a means of detecting HSV. RESULTS: Detection of HSV 1/2 DNA in clinical specimens by real-time PCR did not require that the specimen be subjected to nucleic acid extraction/purification prior to analysis. Each specimen that was detectable by real-time PCR when analyzed in the extracted form was also detectable when analyzed in the unextracted form using the methods herein. The limit of detection of HSV-1 and HSV-2 particles when analyzed in the unextracted form was found to be approximately 17 and 32 virus particles respectively, compared to a sensitivity of 10 copies, for analysis of purified DNA. Omission of the nucleic acid extraction step shortened both the assay time and cost. CONCLUSION: Omission of the nucleic acid extraction step prior to real-time PCR for detection of herpes simplex virus resulted in a more rapid and cost-effective assay, with little impact upon the sensitivity of detection.

Project description:A rapid real-time multiplex PCR assay for detecting and differentiating Bordetella pertussis and Bordetella parapertussis in nasopharyngeal swabs was developed. This assay (LC-PCR-IS) targets the insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively, and is performed using the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.). The analytical sensitivity is less than one organism per reaction. Results for Bordetella culture and/or direct fluorescent antibody testing and a second LightCycler PCR assay (target, pertussis toxin gene) were compared to results of the LC-PCR-IS assay for 111 nasopharyngeal swabs submitted for pertussis testing. Of the specimens, 12 were positive (9 B. pertussis and 3 B. parapertussis) and 68 specimens were negative by all methods. Three other specimens were positive for B. pertussis by at least two of the methods (including the LC-PCR-IS assay), and another 28 specimens were positive for B. pertussis by the LC-PCR-IS assay only. No specimens were negative by the LC-PCR-IS assay and positive by the other methods. A conventional PCR method (target, IS481) was also compared to the LC-PCR-IS assay for a different group of nasopharyngeal swab specimens (n = 96): 44 specimens were positive and 41 specimens were negative for B. pertussis with both PCR methods. Nine specimens were positive for B. pertussis by the LC-PCR-IS assay and negative by the conventional PCR assay, and two specimens were positive for B. pertussis by the conventional PCR assay and negative by the LC-PCR-IS assay. Positivity of the two assays was not significantly different (P = 0.0654). The insertion sequence IS481 is also present in Bordetella holmesii; specimens containing B. holmesii may yield false-positive results. The LC-PCR-IS assay takes approximately 45 min to complete post-nucleic acid extraction, compared to 24 h for the conventional PCR assay previously used in our laboratory. The LC-PCR-IS assay is easier to perform than the conventional PCR assay, and the closed system decreases the chance of contamination. All of these characteristics represent a significant improvement in the detection of B. pertussis and B. parapertussis in nasopharyngeal specimens.

Dataset Information

Sequencing and resolution of amplified herpes simplex virus DNA with intermediate melting curves as genotype 1 or 2 by LightCycler PCR assay.

Publications

Sequencing and resolution of amplified herpes simplex virus DNA with intermediate melting curves as genotype 1 or 2 by LightCycler PCR assay.

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