Comparative Evaluation of Several Gene Targets for Designing a Multiplex-PCR for an Early Diagnosis of Extrapulmonary Tuberculosis

Raj, Ankush; Singh, Netrapal; Gupta, K. B.; Chaudhary, Dhruva; Yadav, Akhilesh Kumar; Chaudhary, Anil; Agarwal, Kshitij; Varma-Basil, Mandira; Prasad, Rajendra; Khuller, G. K.; Mehta, Promod K

doi:10.3349/ymj.2016.57.1.88

Cited by 44 publications

(34 citation statements)

References 34 publications

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“…The pooled sensitivity and AUC of MPB64 LAMP were higher than those of IS6110 LAMP against CRS. This result was consistent with those of studies using other NAATs [ 40 , 41 ]. Included studies using culture as the reference standard were limited, and the difference in pooled sensitivity and specificity for the two target genes could not be analyzed.…”

Section: Discussionsupporting

confidence: 93%

Diagnostic accuracy of the loop-mediated isothermal amplification assay for extrapulmonary tuberculosis: A meta-analysis

Shen

Zhong

et al. 2018

PLoS ONE

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BackgroundLoop-mediated isothermal amplification (LAMP) is used to detect pulmonary tuberculosis (PTB); however, the diagnostic accuracy of the LAMP assay for extrapulmonary tuberculosis (EPTB) is unclear. We performed a meta-analysis to evaluate the performance of LAMP in the detection of EPTB.MethodsWe searched PubMed, EMBASE, the Cochrane Library, China National Knowledge Infrastructure (CNKI), and the Wanfang database for studies published before Sep 16, 2017. We reviewed studies and compared the performance of LAMP with that of a composite reference standard (CRS) and culture for clinically suspected EPTB. We used a bivariate random-effects model to perform meta-analyses and used meta-regression and subgroup analysis to analyze sources of heterogeneity.ResultsFourteen articles including 24 independent studies (16 compared LAMP to CRS, 8 to culture) of EPTB were identified. LAMP showed a pooled sensitivity of 77% (95% confidence interval (CI) 68–85), specificity of 99% (95% CI 96–100), and area under SROC curves (AUC) of 0.96 (95% CI 0.94–0.97) against CRS. It showed a pooled sensitivity of 93% (95% CI 88–96), specificity of 77% (95% CI 64–86), and AUC of 0.94 (95% CI 0.92–0.96) against culture. The pooled sensitivity, specificity, and AUC of MPB64 LAMP were 86% (95% CI 86–86), 100% (95% CI 100–100), and 0.97 (95% CI 0.95–0.98), respectively, and those of IS6110 LAMP were 75% (95% CI 64–84), 99% (95% CI 90–100), and 0.91 (95% CI 0.88–0.93), respectively, compared with CRS.ConclusionsThese results suggest good diagnostic efficacy of LAMP in the detection of EPTB. Additionally, the diagnostic efficacy of MPB64 LAMP was superior to that of IS6110 LAMP.

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Section: Discussionsupporting

confidence: 93%

Diagnostic accuracy of the loop-mediated isothermal amplification assay for extrapulmonary tuberculosis: A meta-analysis

Shen

Zhong

et al. 2018

PLoS ONE

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“…In a comparative study between common diagnostic target sequences of TB, the sensitivity of mpb64 was highest (84% in confirmed cases and 77.5% in clinically suspected cases). The clinical sensitivity of other targets was as follows: mpb64 > IS6110 > hsp65 > 38KDa ( pstS1 ) > 30KDa ( fbpB ) > esat6 > cfp10 > devR [ 5 ].…”

Section: Discussionmentioning

confidence: 99%

“…PCR is the most important molecular diagnostic method used to detect TB during past two decades, and it has maintained its position despite the introduction of newer methods such as loop -mediated isothermal amplification (LAMP). Over the past years, various target sequences were used for TB PCR such as IS6110 , mpb64 , devR , hsp65 , 38 KDa ( pstS1 ), 30 KDa ( fbpB ), esat6 , cfp10 , 16S rDNA gene ( rrs ) and rpoB [ 5 – 9 ]. Diagnostic tests based on these targets have high sensitivity and specificity.…”

Section: Introductionmentioning

confidence: 99%

Modified genome comparison method: a new approach for identification of specific targets in molecular diagnostic tests using Mycobacterium tuberculosis complex as an example

et al. 2018

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BackgroundThe first step of designing any genome-based molecular diagnostic test is to find a specific target sequence. The modified genome comparison method is one of the easiest and most comprehensive ways to achieve this goal. In this study, we aimed to explain this method with the example of Mycobacterium tuberculosis complex and investigate its efficacy in a diagnostic test.MethodsA specific target was identified using modified genome comparison method and an in-house PCR test was designed. To determine the analytical sensitivity and specificity, 10 standard specimens were used. Also, 230 specimens were used to determine the clinical sensitivity and specificity.ResultsThe identity and query cover of our new diagnostic target (5KST) were ≥ 90% with M. tuberculosis complex. The 5KST-PCR sensitivity was 100% for smear-positive, culture-positive and 85.7% for smear-negative, culture-positive specimens. All of 100 smear-negative, culture-negative specimens were negative in 5KST-PCR (100% clinical specificity). Analytical sensitivity of 5KST-PCR was approximately 1 copy of genomic DNA per microliter.ConclusionsModified genome comparison method is a confident way to find specific targets for use in diagnostic tests. Accordingly, the 5KST-PCR designed in this study has high sensitivity and specificity and can be replaced for conventional TB PCR tests.Electronic supplementary materialThe online version of this article (10.1186/s12879-018-3417-x) contains supplementary material, which is available to authorized users.

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“…The multiplex PCR method is increasingly used in animal diagnostics; examples include testing of material for several pathogens simultaneously [18,19]. The approach used in the present study, consisting in the amplifi cation of two regions in the genome of the same pathogen, has been used in many studies confi rming the greater specifi city of multiplex reactions [20][21][22]. The multiplex RT-PCR technique, simultaneously amplifying fragments of the H5, N1 and M genes, has been used to diagnose avian infl uenza, with the sensitivity of the assay specifi ed at 10 3 copies/ul [23].…”

Section: Discussionmentioning

confidence: 92%

Duplex PCR for Detection of Aleutian Disease Virus from Biological and Environmental Samples

2019

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Aleutian disease is one of the most serious disease entities affecting mink farms. The disease causes significant economic losses in mink breeding countries. The aim of the study was to optimize a diagnostic test based on duplex PCR to enable detection of Aleutian disease virus in biological and environmental samples. Blood (n = 40) and spleen (n = 40) samples from animals with suspected infection, and swabs from cages in which infected animals were kept (n = 20) were used for analysis. DNA was isolated from the samples, followed by optimization of the duplex PCR reaction targeting sequences coding NS1 and VP2 proteins. The qPCR method was used to determine the sensitivity of the reaction. The specificity of the analysis was confirmed by the sequencing results. Optimized duplex PCR enabled detection of Aleutian Mink Disease Virus (AMDV) genetic material in biological and environmental samples. Testing of the sensitivity of the method indicated clear amplification for both primer pairs at 102 copies of viral DNA in a reaction. Sequencing confirmed the specificity of the reaction, which in the case of both primer pairs indicated an over 90% agreement between the isolates and the variants of the virus from the databases. The use of duplex PCR to detect two regions of the AMDV genome may increase the sensitivity and specificity of the method and significantly expand the possibilities of further analysis based on sequencing.

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Comparative Evaluation of Several Gene Targets for Designing a Multiplex-PCR for an Early Diagnosis of Extrapulmonary Tuberculosis

Cited by 44 publications

References 34 publications

Diagnostic accuracy of the loop-mediated isothermal amplification assay for extrapulmonary tuberculosis: A meta-analysis

Diagnostic accuracy of the loop-mediated isothermal amplification assay for extrapulmonary tuberculosis: A meta-analysis

Modified genome comparison method: a new approach for identification of specific targets in molecular diagnostic tests using Mycobacterium tuberculosis complex as an example

Duplex PCR for Detection of Aleutian Disease Virus from Biological and Environmental Samples

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