Dataset Information

Multi-Fluorescence Real-Time PCR Assay for Detection of RIF and INH Resistance of M. tuberculosis.

ABSTRACT:

Background

Failure to early detect multidrug-resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes, and highlights the need to rapidly detect resistance to rifampicin (RIF) and isoniazid (INH).

Methods

In Multi-Fluorescence quantitative Real-Time PCR (MF-qRT-PCR) assay, 10 probes labeled with four kinds of fluorophores were designed to detect the mutations in regions of rpoB, katG, mabA-inhA, oxyR-ahpC, and rrs. The efficiency of MF-qRT-PCR assay was tested using 261 bacterial isolates and 33 clinical sputum specimens. Among these samples, 227 Mycobacterium tuberculosis isolates were analyzed using drug susceptibility testing (DST), DNA sequencing and MF-qRT-PCR assay.

Results

Compared with DST, MF-qRT-PCR sensitivity and specificity for RIF-resistance were 94.6 and 100%, respectively. And the detection sensitivity and specificity for INH-resistance were 85.9 and 95.3%, respectively. Compared with DNA sequencing, the sensitivity and specificity of our assay were 97.2 and 100% for RIF-resistance and 97.9 and 96.4% for INH-resistance. Compared with Phenotypic strain identification, MF-qRT-PCR can distinguish 227 M. tuberculosis complexes (MTC) from 34 Non-tuberculous mycobacteria (NTM) isolates with 100% accuracy rate.

Conclusions

MF-qRT-PCR assay was an efficient, accurate, reliable, and easy-operated method for detection of RIF and INH-resistance, and distinction of MTC and NTM of clinical isolates.

SUBMITTER: Peng J

PROVIDER: S-EPMC4850356 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Publications

Multi-Fluorescence Real-Time PCR Assay for Detection of RIF and INH Resistance of M. tuberculosis.

Peng Jingfu J Yu Xiaoli X Cui Zhenling Z Xue Wenfei W Luo Ziyi Z Wen Zilu Z Liu Minghua M Jiang Danqing D Zheng Heping H Wu Hai H Zhang Shulin S Li Yao Y

Frontiers in microbiology 20160429

<h4>Background</h4>Failure to early detect multidrug-resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes, and highlights the need to rapidly detect resistance to rifampicin (RIF) and isoniazid (INH).<h4>Methods</h4>In Multi-Fluorescence quantitative Real-Time PCR (MF-qRT-PCR) assay, 10 probes labeled with four kinds of fluorophores were designed to detect the mutations in regions of rpoB, katG, mabA-inhA, oxyR-ahpC, and rrs. The efficiency of MF-qRT-PCR assay ...[more]

PMID: 27199947

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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.

Project description:IntroductionCalf diarrhea is a significant condition that has a strong effect on the cattle industry, resulting in huge economic losses annually. Bovine torovirus (BToV), bovine enterovirus (BEV), bovine norovirus (BNoV), bovine coronavirus (BCoV), bovine rotavirus (BRV), and bovine viral diarrhea virus (BVDV) are key pathogens that have been implicated in calf diarrhea. Among these viruses, there remains limited research on BToV, BEV, and BNoV, with no available vaccines or drugs for their prevention and control. Although commercial vaccines exist for BCoV, BRV, and BVDV, the prevalence of these diseases remains high.MethodsTo address this issue, we developed a multiplex real-time fluorescence quantitative PCR method for detecting BToV, BEV, BNoV, BCoV, BRV, and BVDV. This method can be used to effectively monitor the prevalence of these six viruses and serve as a reference for future prevention and control strategies. In this study, we specifically designed primers and probes for the BNoV Rdrp, BEV 5'UTR, BToV M, BCoV N, BRV NSP5, and BVDV 5'UTR genes.ResultsThis method was determined to be efficient, stable, and sensitive. The lowest detectable levels of plasmids for BNoV, BEV, BToV, BRV, BCoV, and BVDV were 1.91 copies/μL, 96.0 copies/μL, 12.8 copies/μL, 16.4 copies/μL, 18.2 copies/μL, and 65.3 copies/μL, respectively. Moreover, the coefficients of variation for all six detection methods were < 3%; they also exhibited a strong linear relationship (R2 ≥ 0.98), and an amplification efficiency of 90%-110%. A total of 295 fecal and anal swabs were collected from calves with diarrhea in Guangdong, China. The positive rates for BToV, BEV, BNoV, BCoV, BR, and BVDV were determined to be 0.34% (1/295), 6.10% (18/295), 0.68% (2/295), 1.36% (4/295), 10.85% (32/295), and 2.03% (6/295), respectively. Notably, BEV and BRV exhibited the highest prevalence.DiscussionAdditionally, this study identified the occurrence of BToV and BNoV in Guangdong for the first time. In summary, this study successfully established an effective method for detecting several important bovine viruses; ultimately, this holds strong implications for the future development of the cattle industry.

Dataset Information

Multi-Fluorescence Real-Time PCR Assay for Detection of RIF and INH Resistance of M. tuberculosis.

Background

Methods

Results

Conclusions

Publications

Multi-Fluorescence Real-Time PCR Assay for Detection of RIF and INH Resistance of M. tuberculosis.

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