Diagnostic potential of multi‐targeted LAMP (loop‐mediated isothermal amplification) for osteoarticular tuberculosis

Huang

et al. 2017

Sci Rep

103

This study established a constant-temperature fluorescence quantitative detection method, combining loop-mediated isothermal amplification (LAMP) with molecular beacons. The advantages of LAMP are its convenience and efficiency, as it does not require a thermocycler and results are easily visualized by the naked eye. However, a major disadvantage of current LAMP techniques is the use of indirect evaluation methods (e.g., electrophoresis, SYBR Green I dye, precipitation, hydroxynaphthol blue dye, the turbidimetric method, calcein/Mn2+ dye, and the composite probe method), which cannot distinguish between the desired products and products of nonspecific amplification, thereby leading to false positives. Use of molecular beacons avoids this problem because molecular beacons produce fluorescence signals only when binding to target DNA, thus acting as a direct indicator of amplification products. Our analyses determined the optimal conditions for molecular beacons as an evaluation tool in LAMP: beacon length of 25–45 bp, beacon concentration of 0.6–1 pmol/μL, and reaction temperature of 60–65 °C. In conclusion, we validated a novel molecular beacon loop-mediated isothermal amplification method (MB-LAMP), realizing the direct detection of LAMP product.

mentioning

confidence: 99%

Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay

Huang

et al. 2017

Sci Rep

103

“…Eight articles used only one type of specimen (e.g., only CSF) [ 20 – 22 , 24 – 28 ]. The other studies used multiple types of specimens [ 15 – 19 , 23 ]. Only one study provided HIV infection status [ 26 ].…”

Section: Resultsmentioning

confidence: 99%

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

Shen

Zhong

et al. 2018

PLoS ONE

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.

“…The IS6110 gene has been widely applied for PCR detections in the M. tuberculosis complex genome, which belongs to a family of IS of the IS3 category with high speci city [22]. However, some of M. tuberculosis strains absence of IS6110 gene were found in several clinical investigations during recent decades [23,24]. Therefore, in this research, we added another target gene, the mpb64 gene.…”

Section: Discussionmentioning

confidence: 99%

Two Target Genes Based Multiple Cross Displacement Amplification Combined with Lateral Flow Biosensor for the Detection of Mycobacrterium Tuberculosis Complex

Huang

Xiao

Yang

et al. 2020

Preprint

Background: Mycobacterium tuberculosis complex (MTBC) causes tuberculosis (TB), which is a global public health problem that seriously endangers public health. Hence, development of a new and rapid method to detect MTBC is of great significance for prevention and treatment of TB. Results: In this study, a multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor (MCDA-LFB) was developed to simultaneously detect two target genes (IS6110 and mpb64) of MTBC. One suit of specific multiple cross displacement amplification (MCDA) primers, which was designed for IS6110 and mpb64 gene, respectively, was validated through using the genomic DNA extracted from reference strain H37Rv. The MCDA products were analyzed using real-time turbidity curve, colorimetric indicator (Malachite Green, MG) and LFB. The conditions of amplification temperature and time were optimized and the established MCDA-LFB method was applied to detect the sputum specimens and MTBC strains from clinical samples. The results show that two sets of MCDA primers for the IS6110 and mpb64 genes have detected MTBC validly. The MCDA reaction conditions were optimized at 67 °C for 35 min. The limit of detection of MCDA assay based on IS6110 and mpb64 genes was 100 fg of genomic DNA template per reaction. The specificity of MCDA-LFB detection was 100%, and no cross-reactions for non-MTBC strains detection. The positive rate of MCDA-LFB for the detection of MTBC strains was equal to that of semi-nested automatic real-time PCR (Xpert MTB/RIF), and had a higher positive rate than acid-fast staining (AFS) when used for the detection of sputum samples. The whole procedure of MCDA-LFB, including genomic template preparation, MCDA reaction and products analysis, was completed with 70 min.Conclusion: The simplicity, rapidity, sensitivity and reliability of the MCDA-LFB based on IS6110 and mpb64 gene of MTBC developed in this study make it potentially significant for the prevention and treatment of TB.