Development of a nested qualitative real-time PCR assay to detect Aspergillus species DNA in clinical specimens.
Ontology highlight
ABSTRACT:
SUBMITTER: Halliday C
PROVIDER: S-EPMC1248492 | biostudies-literature | 2005 Oct
REPOSITORIES: biostudies-literature
Publications
Development of a nested qualitative real-time PCR assay to detect Aspergillus species DNA in clinical specimens.
Halliday Catriona C Wu Qi Xuan QX James Gregory G Sorrell Tania T
Journal of clinical microbiology 20051001 10
We adapted a nested Aspergillus PCR to a nested qualitative real-time format on a Light Cycler system. An evaluation using 134 clinical specimens showed that the real-time PCR assay significantly reduced the time for results to be made available without compromising sensitivity and was less labor-intensive. Its use will aid in the rapid diagnosis of invasive aspergillosis. ...[more]
Similar Datasets
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:The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) is a multilateral environmental agreement to ensure that the international trade of threatened species is either prohibited (Appendix I listed species) or being conducted legally, sustainably, and transparently (Appendix II listed species). Twelve threatened shark species exploited for their fins, meat, and other products have been listed under CITES Appendix II. Sharks are often traded in high volumes, some of their products are visually indistinguishable, and most importing/exporting nations have limited capacity to detect illicit trade and enforce the regulations. High volume shipments often must be screened after only a short period of detainment (e.g., a maximum of 24?hours), which together with costs and capacity issues have limited the use of DNA approaches to identify illicit trade. Here, we present a reliable, field-based, fast (<4?hours), and cost effective ($0.94 USD per sample) multiplex real-time PCR protocol capable of detecting nine of the twelve sharks listed under CITES in a single reaction. This approach facilitates detection of illicit trade, with positive results providing probable cause to detain shipments for more robust forensic analysis. We also provide evidence of its application in real law enforcement scenarios in Hong Kong. Adoption of this approach can help parties meet their CITES requirements, avoiding potential international trade sanctions in the future.
| S-EPMC6218538 | biostudies-literature
Project description:A novel multitarget real-time PCR (RT-PCR) assay for the rapid identification of Bordetella pertussis, B. parapertussis, and B. holmesii was developed using multicopy insertion sequences (ISs) in combination with the pertussis toxin subunit S1 (ptxS1) singleplex assay. The RT-PCR targets for the multiplex assay include IS481, commonly found in B. pertussis and B. holmesii; IS1001 of B. parapertussis; and the IS1001-like sequence of B. holmesii. Overall, 402 Bordetella species and 66 non-Bordetella species isolates were tested in the multitarget assay. Cross-reactivity was found only with 5 B. bronchiseptica isolates, which were positive with IS1001 of B. parapertussis. The lower limit of detection (LLOD) of the multiplex assay was similar to the LLOD of each target in an individual assay format, which was approximately 1 genomic equivalent per reaction for all targets. A total of 197 human clinical specimens obtained during cough-illness outbreak investigations were used to evaluate the multitarget RT-PCR assay. The multiplex assay results from 87 clinical specimens were compared to the individual RT-PCR assay and culture results. The multitarget assay is useful as a diagnostic tool to confirm B. pertussis infections and to rapidly identify other Bordetella species. In conclusion, the use of this multitarget RT-PCR approach increases specificity, while it decreases the amount of time, reagents, and specimen necessary for RT-PCRs used for accurate diagnosis of pertussis-like illness.
| S-EPMC3232951 | biostudies-literature
Project description:The polymerase chain reaction (PCR) is widely used as a diagnostic tool in clinical laboratories and is particularly effective for detecting and identifying infectious agents for which routine culture and microscopy methods are inadequate. Invasive fungal disease (IFD) is a major cause of morbidity and mortality in immunosuppressed patients, and optimal diagnostic criteria are contentious. Although PCR-based methods have long been used for the diagnosis of invasive aspergillosis (IA), variable performance in clinical practice has limited their value. This shortcoming is a consequence of differing sample selection, collection and preparation protocols coupled with a lack of standardisation of the PCR itself. Furthermore, it has become clear that the performance of PCR-based assays in general is compromised by the inadequacy of experimental controls, insufficient optimisation of assay performance as well as lack of transparency in reporting experimental details. The recently published "Minimum Information for the publication of real-time Quantitative PCR Experiments" (MIQE) guidelines provide a blueprint for good PCR assay design and unambiguous reporting of experimental detail and results. We report the first real-time quantitative PCR (qPCR) assay targeting Aspergillus species that has been designed, optimised and validated in strict compliance with the MIQE guidelines. The hydrolysis probe-based assay, designed to target the 18S rRNA DNA sequence of Aspergillus species, has an efficiency of 100% (range 95-107%), a dynamic range of at least six orders of magnitude and limits of quantification and detection of 6 and 0.6 Aspergillus fumigatus genomes, respectively. It does not amplify Candida, Scedosporium, Fusarium or Rhizopus species and its clinical sensitivity is demonstrated in histological material from proven IA cases, as well as concordant PCR and galactomannan data in matched broncho-alveolar lavage and blood samples. The robustness, specificity and sensitivity of this assay make it an ideal molecular diagnostic tool for clinical use.
| S-EPMC3393739 | biostudies-literature
Project description:Bifidobacterium species are used as probiotics to provide beneficial effects to humans. These effects are specific to some species or subspecies of Bifidobacterium. However, some Bifidobacterium species or subspecies are not distinguished because similarity of 16S rRNA and housekeeping gene sequences within Bifidobacterium species is very high. In this study, we developed a real-time polymerase chain reaction (PCR) assay to rapidly and accurately detect 22 Bifidobacterium species by selecting genetic markers using comparative genomic analysis. A total of 210 Bifidobacterium genome sequences were compared to select species- or subspecies-specific genetic markers. A phylogenetic tree based on pan-genomes generated clusters according to Bifidobacterium species or subspecies except that two strains were not grouped with their subspecies. Based on pan-genomes constructed, species- or subspecies-specific genetic markers were selected. The specificity of these markers was confirmed by aligning these genes against 210 genome sequences. Real-time PCR could detect 22 Bifidobacterium specifically. We constructed the criterion for quantification by standard curves. To further test the developed assay for commercial food products, we monitored 26 probiotic products and 7 dairy products. Real-time PCR results and labeling data were then compared. Most of these products (21/33, 63.6%) were consistent with their label claims. Some products labeled at species level only can be detected up to subspecies level through our developed assay.
| S-EPMC7493681 | biostudies-literature
Project description:Coccidioides spp. are dimorphic fungal pathogens endemic to the semiarid regions of North, Central, and South America. Currently, direct smear and culture are the most common means of identifying Coccidioides spp. While these methods offer relatively sensitive and specific means of detecting Coccidioides spp., growth in culture may take up to 3 weeks, potentially delaying the diagnosis and initiation of appropriate antifungal therapy. In addition, growth of the organism represents a significant safety risk to laboratory personnel. The need for a rapid and safe means of diagnosing coccidioidomycosis prompted us to develop a real-time PCR assay to detect Coccidioides spp. directly from clinical specimens. Primers and fluorescent resonance energy transfer (FRET) probes were designed to target the internal transcribed spacer 2 region of Coccidioides. The assay's limit of detection is below 50 targets per reaction. An analysis of 40 Coccidioides sp. clinical isolates grown in culture demonstrated 100% sensitivity of the assay. A cross-reactivity panel containing fungi, bacteria, mycobacteria, and viruses was tested and demonstrated 100% specificity for Coccidioides spp. An analysis of 266 respiratory specimens by LightCycler PCR demonstrated 100% sensitivity and 98.4% specificity for Coccidioides spp. compared with culture. Analysis of 66 fresh tissue specimens yielded 92.9% sensitivity and 98.1% specificity versus those of the culture method. The sensitivity of the assay testing 148 paraffin-embedded tissue samples is 73.4%. A rapid method for the detection of Coccidioides spp. directly from clinical material will greatly assist in the timely diagnosis and treatment of patients, while at the same time decreasing the risk of accidental exposure to laboratory personnel.
| S-EPMC1828991 | biostudies-literature
Project description:Bartonella endocarditis is a severe disease for which blood cultures frequently remain negative. We tested three PCR assays by using specimens of serum sampled early during the disease from 43 patients diagnosed in our laboratory as having Bartonella endocarditis on the basis of serological, culture, and/or valvular molecular detection. We tested a two-step nested PCR (TSN-PCR), a one-step nested PCR (OSN-PCR) with a regular thermal cycler, and a one-step nested PCR with the LightCycler (LCN-PCR). These assays were performed with primers derived from the riboflavin synthase-encoding gene ribC, never before amplified in our laboratory. Due to contamination of negative controls, the results of the TSN-PCR were not interpretable, and this technique was no longer considered. The LCN-PCR had a specificity of 100% and a sensitivity of 58.1%, higher than those of the OSN-PCR (18.6%; P < 0.01) and prolonged blood culturing (7.1%; P < 0.01). The LCN-PCR results correlated strictly with those of other direct diagnostic tests, when available, and identified the causative species for six patients previously diagnosed on the basis of serological analysis only. The efficacy of the LCN-PCR was not influenced by antibiotics (P = 0.96) but was altered by prolonged storage of serum specimens at -20 degrees C (P = 0.04). Overall, the LCN-PCR is specific and more sensitive than traditional methods (i.e., culturing and/or PCR with EDTA-treated blood). It can easily be applied to the diagnosis of patients with suspected Bartonella endocarditis, especially when only serum is available.
| S-EPMC150267 | biostudies-literature
Project description:The diagnosis of tuberculous meningitis (TBM) remains a complex issue because the most widely used conventional diagnostic tools, such as culture and PCR assay for cerebrospinal fluid (CSF) samples, are unable to rapidly detect Mycobacterium tuberculosis with sufficient sensitivity in the acute phase of TBM. Based on TaqMan PCR, we designed a novel technique consisting of an internally controlled quantitative nested real-time (QNRT) PCR assay that provided a marked improvement in detection sensitivity and quantification. We applied this novel technique to quantitatively detect M. tuberculosis DNA in CSF samples from patients with suspected TBM. For use as the internal control in the measurement of the M. tuberculosis DNA copy numbers in the QNRT-PCR assay, the original mutation (M) plasmid, which included an artificial random 22-nucleotide sequence within an inserted DNA fragment of the MPB64 gene of M. tuberculosis, was prepared. The QNRT-PCR assay showed high sensitivity and specificity that were approximately equivalent to those of the conventional nested PCR assay. Moreover, the QNRT-PCR assay made it possible to precisely and quantitatively detect the initial copy number of M. tuberculosis DNA in CSF samples. Therefore, compared to the conventional PCR assay, the QNRT-PCR assay can be considered a more useful and advanced technique for the rapid and accurate diagnosis of TBM. To establish the superiority of this novel technique in TBM diagnosis, it will be necessary to accumulate data from a larger number of patients with suspected TBM.
| S-EPMC1393095 | biostudies-literature
Project description:The mumps virus is a negative-strand RNA virus in the family Paramyxoviridae. Mumps infection results in an acute illness with symptoms including fever, headache, and myalgia, followed by swelling of the salivary glands. Complications of mumps can include meningitis, deafness, pancreatitis, orchitis, and first-trimester abortion. Laboratory confirmation of mumps infection can be made by the detection of immunoglobulin M-specific antibodies to mumps virus in acute-phase serum samples, the isolation of mumps virus in cell culture, or by detection of the RNA of the mumps virus by reverse transcription (RT)-PCR. We developed and validated a multiplex real-time RT-PCR assay for rapid mumps diagnosis in a clinical setting. This assay used oligonucleotide primers and a TaqMan probe targeting the mumps SH gene, as well as primers and a probe that targeted the human RNase P gene to assess the presence of PCR inhibitors and as a measure of specimen quality. The test was specific, since it did not amplify a product from near-neighbor viruses, as well as sensitive and accurate. Real-time RT-PCR results showed 100% correlation with results from viral culture, the gold standard for mumps diagnostic testing. Assay efficiency was over 90% and displayed good precision after performing inter- and intraassay replicates. Thus, we have developed and validated a molecular method for rapidly diagnosing mumps infection that may be used to complement existing techniques.
| S-EPMC2045251 | biostudies-literature
Project description:Real-time PCR, using dual-labeled fluorescent probes targeting the beta-giardin gene, was used to detect Giardia lamblia in human stool specimens and to discriminate between isolates from the two major genetic assemblages of G. lamblia infective to humans, assemblages A and B.
| S-EPMC446313 | biostudies-literature