Dataset Information

Rapid diagnosis of Mycoplasma pneumonia infection by denaturation bubble-mediated strand exchange amplification: comparison with LAMP and real-time PCR.

ABSTRACT: M. pneumoniae infection is often ignored due to its similar clinical symptom with respiratory tract infections caused by bacteria or viruses, and thus leading to misdiagnosis and delayed treatment. It is critical to develop a rapid, sensitive and specific diagnosis method. Denaturation Bubble-mediated Strand Exchange Amplification (SEA) was established, which is an isothermal method with only a primer pair and one Bst DNA polymerase. Notably, colorimetric SEA assay was developed with simple visual readout, making instrument-independent in detection step. The method could detect as low as 1.0?×?104 copies/mL genomic DNA within 60?min. Considering that more than 80% infected patients have 1.0?×?105-1.0?×?107 copies/mL M. pneumonia DNA, SEA is available for the practical diagnosis of M. pneumoniae in clinical specimens. Through comparing 224 sputum specimens, excellent performance of SEA assay with 90.48% sensitivity and 100% specificity relative to real-time PCR was observed. Compared with LAMP, a comparable sensitivity and low false positive rate was observed for SEA method. Therefore, SEA is a promising method for detecting M. pneumoniae directly from clinical specimens, which is especially suitable for point-of-care testing in primary care facilities and resource-limited settings with minimal equipment and technological expertises.

SUBMITTER: Shi W

PROVIDER: S-EPMC6351683 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Rapid diagnosis of Mycoplasma pneumonia infection by denaturation bubble-mediated strand exchange amplification: comparison with LAMP and real-time PCR.

Shi Wenqiang W Wei Manman M Wang Qing Q Wang Hongwei H Ma Cuiping C Shi Chao C

Scientific reports 20190129 1

M. pneumoniae infection is often ignored due to its similar clinical symptom with respiratory tract infections caused by bacteria or viruses, and thus leading to misdiagnosis and delayed treatment. It is critical to develop a rapid, sensitive and specific diagnosis method. Denaturation Bubble-mediated Strand Exchange Amplification (SEA) was established, which is an isothermal method with only a primer pair and one Bst DNA polymerase. Notably, colorimetric SEA assay was developed with simple visu ...[more]

PMID: 30696841

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Project description:BackgroundContagious Bovine Pleuropneumonia (CBPP) is the most important chronic pulmonary disease of cattle on the African continent causing severe economic losses. The disease, caused by infection with Mycoplasma mycoides subsp. mycoides is transmitted by animal contact and develops slowly into a chronic form preventing an early clinical diagnosis. Because available vaccines confer a low protection rate and short-lived immunity, the rapid diagnosis of infected animals combined with traditional curbing measures is seen as the best way to control the disease. While traditional labour-intensive bacteriological methods for the detection of M. mycoides subsp. mycoides have been replaced by molecular genetic techniques in the last two decades, these latter approaches require well-equipped laboratories and specialized personnel for the diagnosis. This is a handicap in areas where CBPP is endemic and early diagnosis is essential.ResultsWe present a rapid, sensitive and specific diagnostic tool for M. mycoides subsp. mycoides detection based on isothermal loop-mediated amplification (LAMP) that is applicable to field conditions. The primer set developed is highly specific and sensitive enough to diagnose clinical cases without prior cultivation of the organism. The LAMP assay detects M. mycoides subsp. mycoides DNA directly from crude samples of pulmonary/pleural fluids and serum/plasma within an hour using a simple dilution protocol. A photometric detection of LAMP products allows the real-time visualisation of the amplification curve and the application of a melting curve/re-association analysis presents a means of quality assurance based on the predetermined strand-inherent temperature profile supporting the diagnosis.ConclusionThe CBPP LAMP developed in a robust kit format can be run on a battery-driven mobile device to rapidly detect M. mycoides subsp. mycoides infections from clinical or post mortem samples. The stringent innate quality control allows a conclusive on-site diagnosis of CBPP such as during farm or slaughter house inspections.

Project description:BACKGROUND:DNA genotyping with mutation-specific TaqMan(R) probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations. METHODS:Minor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature (T(c)) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the T(c), followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors. RESULTS:COLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors. CONCLUSIONS:The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.

Dataset Information

Rapid diagnosis of Mycoplasma pneumonia infection by denaturation bubble-mediated strand exchange amplification: comparison with LAMP and real-time PCR.

Publications

Rapid diagnosis of Mycoplasma pneumonia infection by denaturation bubble-mediated strand exchange amplification: comparison with LAMP and real-time PCR.

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