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Real-time PCR assay to detect smallpox virus.

ABSTRACT: We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA from 48 different isolates of variola virus; 25 different strains and isolates of camelpox, cowpox, ectromelia, gerbilpox, herpes, monkeypox, myxoma, rabbitpox, raccoonpox, skunkpox, vaccinia, and varicella-zoster viruses; and two rickettsial species at concentrations mostly ranging from 100 fg/ microl to 1 ng/ microl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/ microl to 1 ng/ microl. On the Smart Cycler platform, 2 samples with false-positive results were detected among the 116 samples not containing variola virus tested; i.e., the overall specificity of the assay was 98.3%. On the LightCycler platform, five samples with false-positive results were detected (overall specificity, 95.7%). Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/ microl to 1 ng/ microl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform. Thus, the clinical sensitivities were 96.1% for the Smart Cycler instrument and 99.5% for the LightCycler instrument. The vast majority of these samples were derived from virus-infected cell cultures and variola virus-infected tissues; thus, the DNA material contained both viral DNA and cellular DNA. Of the 43 samples that contained purified variola virus DNA ranging in concentration from 1 fg/ microl to 1 ng/ microl, the assay correctly detected the virus in all 43 samples on both the Smart Cycler and the LightCycler platforms. The assay may be useful for the early detection of smallpox virus infections should such infections occur as a result of a deliberate or an accidental recurrence.

SUBMITTER: Sofi Ibrahim M

PROVIDER: S-EPMC179856 | biostudies-literature | 2003 Aug

REPOSITORIES: biostudies-literature

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Real-time PCR assay to detect smallpox virus.

Sofi Ibrahim M M Kulesh David A DA Saleh Sharron S SS Damon Inger K IK Esposito Joseph J JJ Schmaljohn Alan L AL Jahrling Peter B PB

Journal of clinical microbiology 20030801 8

We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA fr ...[more]

PMID: 12904397

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Project description:A screening assay for real-time LightCycler (Roche Applied Science, Mannheim, Germany) PCR identification of smallpox virus DNA was developed and compiled in a kit system under good manufacturing practice conditions with standardized reagents. In search of a sequence region unique to smallpox virus, the nucleotide sequence of the 14-kDa fusion protein gene of each of 14 variola virus isolates of the Russian World Health Organization smallpox virus repository was determined and compared to published sequences. PCR primers were designed to detect all Eurasian-African species of the genus ORTHOPOXVIRUS: A single nucleotide mismatch resulting in a unique amino acid substitution in smallpox virus was used to design a hybridization probe pair with a specific sensor probe that allows reliable differentiation of smallpox virus from other orthopoxviruses by melting-curve analysis. The applicability was demonstrated by successful amplification of 120 strains belonging to the orthopoxvirus species variola, vaccinia, camelpox, mousepox, cowpox, and monkeypox virus. The melting temperatures (T(m)s) determined for 46 strains of variola virus (T(m)s, 55.9 to 57.8 degrees C) differed significantly (P = 0.005) from those obtained for 11 strains of vaccinia virus (T(m)s, 61.7 to 62.7 degrees C), 15 strains of monkeypox virus (T(m)s, 61.9 to 62.2 degrees C), 40 strains of cowpox virus (T(m)s, 61.3 to 63.7 degrees C), 8 strains of mousepox virus (T(m), 61.9 degrees C), and 8 strains of camelpox virus (T(m)s, 64.0 to 65.0 degrees C). As most of the smallpox virus samples were derived from infected cell cultures and tissues, smallpox virus DNA could be detected in a background of human DNA. By applying probit regression analysis, the analytical sensitivity was determined to be 4 copies of smallpox virus target DNA per sample. The DNAs of several human herpesviruses as well as poxviruses other than orthopoxviruses were not detected by this method. The assay proved to be a reliable technique for the detection of orthopoxviruses, with the advantage that it can simultaneously identify variola virus.

Project description:BACKGROUND: Schistosomiasis constitutes a major public health problem, and 200 million people are estimated to be infected with schistosomiasis worldwide. In Brazil, schistosomiasis has been reported in 19 states, showing areas of high and medium endemicity and a wide range of areas of low endemicity (ALE). Barra Mansa in Rio de Janeiro state has an estimated prevalence of 1%. ALE represent a new challenge for the helminth control because about 75% of infected individuals are asymptomatic and infections occur with a low parasite load (<100 eggs per gram of feces), causing a decrease in sensitivity of stool parasitological techniques, which are a reference for the laboratory diagnosis of this helminth. The objective of this study was to evaluate the performance of a TaqMan quantitative polymerase chain reaction (qPCR) technique in serum and feces DNA samples using the techniques of Kato-Katz (KK), Hoffman, Pons and Janer (HH) as references, during an epidemiological survey using fecal samples and sera from randomized residents from an ALE. METHODS: A cross-sectional study conducted from April to December 2011 using a probabilistic sampling that collected 572 fecal and serum samples. The laboratory diagnostic techniques used were: KK, HH and qPCR (feces and serum). RESULTS: We obtained the following results using the different diagnostic techniques: KK and HH, 0.9% (n =5); qPCR-feces, 9.6% (n =55); and qPCR-serum, 1.4% (n =8). The qPCR-feces presented the highest positivity, whereas the techniques of HH and KK were the least sensitive to detect infections (0.8%). Compared to HH and KK, qPCR-feces showed a statistically significant difference in positivity (p <0.05), although with poor agreement. CONCLUSION: The positivity rate presented by the qPCR approach was far higher than that obtained by parasitological techniques. The lack of adequate surveillance in ALE of schistosomiasis indicates a high possibility of these areas being actually of medium and high endemicity. This study presents a control perspective, pointing to the possibility of using combined laboratory tools in the diagnosis of schistosomiasis in ALE.

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Real-time PCR assay to detect smallpox virus.

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Real-time PCR assay to detect smallpox virus.

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