Detection of Smallpox Virus DNA by LightCycler PCR
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ABSTRACT:
SUBMITTER: Espy M
PROVIDER: S-EPMC139727 | biostudies-literature | 2002 Nov
REPOSITORIES: biostudies-literature
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Project description:Anthrax is a zoonotic disease that is also well recognized as a potential agent of bioterrorism. Routine culture and biochemical testing methods are useful for the identification of Bacillus anthracis, but a definitive identification may take 24 to 48 h or longer and may require that specimens be referred to another laboratory. Virulent isolates of B. anthracis contain two plasmids (pX01 and pX02) with unique targets that allow the rapid and specific identification of B. anthracis by PCR. We developed a rapid-cycle real-time PCR detection assay for B. anthracis that utilizes the LightCycler instrument (LightCycler Bacillus anthracis kit; Roche Applied Science, Indianapolis, Ind.). PCR primers and probes were designed to identify gene sequences specific for both the protective antigen (plasmid pX01) and the encapsulation B protein (plasmid pX02). The assays (amplification and probe confirmation) can be completed in less than 1 h. The gene encoding the protective antigen (pagA) was detected in 29 of 29 virulent B. anthracis strains, and the gene encoding the capsular protein B (capB) was detected in 28 of 29 of the same strains. Three avirulent strains containing only pX01 or pX02, and therefore only pagA or pagB genes, could be detected and differentiated from virulent strains. The assays were specific for B. anthracis: the results were negative for 57 bacterial strains representing a broad range of organisms, including Bacillus species other than anthracis (n = 31) and other non-Bacillus species (n = 26). The analytical sensitivity demonstrated with target DNA cloned into control plasmids was 1 copy per microl of sample. The LightCycler Bacillus anthracis assay appears to be a suitable method for rapid identification of cultured isolates of B. anthracis. Additional clinical studies are required to determine the usefulness of this test for the rapid identification of B. anthracis directly from human specimens.
| S-EPMC120654 | biostudies-literature
Project description:Detection of human cytomegalovirus (CMV) DNA in clinical specimens is considered a cornerstone in the diagnosis of CMV disease. The aim of this study was to evaluate a newly designed LightCycler-based quantitative CMV PCR. Specimens of human origin (n = 200) were tested using the LightCycler PCR, the quantitative COBAS AMPLICOR CMV MONITOR (CACM) assay, and a qualitative in-house PCR assay for the presence of CMV DNA. Samples that were reactive in at least two of the three assays were considered CMV DNA positive (n = 95 [47. 5%]), while samples that were nonreactive in two of the three assays were considered CMV DNA negative (n = 105 [52.5%]). Using the LightCycler assay, CMV DNA was detected in 91 of the 95 CMV DNA-positive human specimens (sensitivity, 95.8%; 95% confidence interval [CI], 89.6 to 98.8) and in 1 of the CMV DNA-negative specimens (specificity, 99%; 95% CI, 94.8 to 99.8). Results of CMV load determination as assessed by both quantitative test systems were correlated (r = 0.73; P < 0.0001; 95% CI, 0.61 to 0.81). Results for undiluted samples containing a high CMV load were more accurate with the LightCycler test than were results obtained with the CACM test, which underestimated the viral load of samples containing high DNA copy numbers. The high level of sensitivity, specificity, accuracy, and rapidity provided by the LightCycler technology are favorable for the use of this system in the detection of CMV DNA in clinical specimens.
| S-EPMC87533 | biostudies-literature
Project description:Laboratory diagnosis of human respiratory syncytial virus (hRSV) infections has traditionally been performed by virus isolation in cell culture and the direct fluorescent-antibody assay (DFA). Reverse transcriptase PCR (RT-PCR) is now recognized as a sensitive and specific alternative for detection of hRSV in respiratory samples. Using the LightCycler instrument, we developed a rapid RT-PCR assay for the detection of hRSV (the LC-RT-PCR) with a pair of hybridization probes that target the hRSV L gene. In the present study, 190 nasopharyngeal aspirate samples from patients with clinically recognized respiratory tract infections were examined for hRSV. The results were then compared to the results obtained with a testing algorithm that combined DFA and a culture-augmented DFA (CA-DFA) assay developed in our laboratory. hRSV was detected in 77 (41%) specimens by LC-RT-PCR and in 75 (39%) specimens by the combination of DFA and CA-DFA. All specimens that were positive by the DFA and CA-DFA testing algorithm were positive by the LC-RT-PCR. The presence of hRSV RNA in the two additional LC-RT-PCR-positive specimens was confirmed by a conventional RT-PCR method that targets the hRSV N gene. The sensitivity of LC-RT-PCR was 50 PFU/ml; and this, together with its high specificity and rapid turnaround time, makes the LC-RT-PCR suitable for the detection of hRSV in clinical specimens.
| S-EPMC154614 | biostudies-literature
Project description: Not available
| S-EPMC88288 | biostudies-literature
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.
| S-EPMC404623 | biostudies-literature
Project description:In order to differentiate species within the Borrelia burgdorferi sensu lato complex, LightCyler PCR and melting-curve analysis of the amplicons of two genes with intraspecies variability, the p66 gene and the recA gene, were performed. It was demonstrated that nested LightCycler PCR amplification of p66 is more sensitive in the detection of borrelia DNA than amplification of the recA gene. B. burgdorferi sensu stricto could be differentiated from Borrelia garinii and Borrelia afzelii by melting-curve analysis of the p66 gene amplicon. B. garinii could be differentiated from B. afzelii and B. burgdorferi sensu stricto by melting-curve analysis of the recA gene amplicon. Therefore, the PCRs complement each other in subtyping different Borrelia species, and combined LightCycler PCR and melting-curve analysis of both target genes is a rapid method to distinguish the three species of B. burgdorferi sensu lato.
| S-EPMC88206 | biostudies-literature
Project description: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.
| S-EPMC179856 | biostudies-literature
Project description:BACKGROUND:Prompt laboratory diagnosis of Herpes simplex virus (HSV) infection facilitates patient management and possible initiation of antiviral therapy. In our laboratory, which receives various specimen types for detection of HSV, we use enzyme immunoassay (EIA) for rapid detection and culture of this virus. The culture of HSV has traditionally been accepted as the diagnostic 'gold standard'. In this study, we compared the use of real time PCR (LightCycler) for amplification, detection and subtyping of specific DNA with our in-house developed rapid and culture tests for HSV. RESULTS:The LightCycler PCR (LC-PCR) detected and subtyped HSV in 99% (66/67) of HSV positive specimens, compared to 81% (54/67) by rapid antigen EIA or 57% (36/63) by culture. A specimen was considered positive when two or more tests yielded HSV identifications or was culture positive. Discordant results were confirmed with an in-house developed PCR-ELISA or DNA sequence analysis. The typing results obtained with the LC-PCR and by culture amplified test were completely concordant. CONCLUSIONS:This study showed that the LC-PCR provided a highly sensitive test for simultaneous detection and subtyping of HSV in a single reaction tube. In addition to increased sensitivity, the LightCycler PCR provided reduced turn-around-times (2 hours) when compared to enzyme immunoassay (4 hours) or culture (4 days).
| S-EPMC116582 | biostudies-literature
Project description:We have developed a fluorescence resonance energy transfer (FRET)-based assay to detect ciprofloxacin resistant (Cp(r)) mutants of the biothreat agent Yersinia pestis. We selected spontaneous mutants of the attenuated Y. pestis KIM 5 strain that were resistant to a ciprofloxacin (CIP) concentration of at least 1 microg/ml. DNA sequencing of gyrA encoded by 65 of these mutants revealed that all isolates contained one of four different point mutations within the quinolone resistance-determining region of gyrA. We developed a FRET-based assay that detected all of these mutations by using a single pair of fluorescent probes with sequences complementary to the wild-type Y. pestis gyrA sequence. Melting peak analysis revealed that the probe-PCR product hybrid was less stable when amplification occurred from any of the four mutant templates. This instability resulted in the PCR product obtained from the Cp(r) Y. pestis strains displaying a 4 to 11 degrees C shift in probe melting temperature. Following optimization of the reaction conditions, we were able to detect approximately 10 pg of purified wild-type template DNA or the presence of approximately 4 CFU of wild-type Y. pestis KIM 5 or Cp(r) mutants in crude lysates. Taken together, our results demonstrate the utility of FRET-based assays for detection of Cp(r) mutants of Y. pestis. This method is both sensitive and rapid.
| S-EPMC88402 | biostudies-literature
Project description:We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3'-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 x 10(7), 1.24 x 10(5), 1.24 x 10(3), and 1.24 x 10(1) genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs. The Smart Cycler was capable of detecting 92.3% of the orthopox virus DNAs and between 75 and 93.8% of the variola virus DNAs. However, all five assays had nearly 100% sensitivity on both machines with samples above the LOD (>12 gene copies). These real-time PCR assays represent a battery of tests to screen for and confirm the presence of variola virus DNA. The early detection of a smallpox outbreak is crucial whether the incident is an act of bioterrorism or an accidental occurrence.
| S-EPMC344443 | biostudies-literature