scholarly journals Development and Preliminary Application of Multiplex Loop-Mediated Isothermal Amplification Coupled With Lateral Flow Biosensor for Detection of Mycobacterium tuberculosis Complex

2021 ◽  
Vol 11 ◽  
Author(s):  
Xingyun Wang ◽  
Guirong Wang ◽  
Yacui Wang ◽  
Shuting Quan ◽  
Hui Qi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Tuberculosis Complex ◽  
Fluorescein Isothiocyanate ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Tuberculosis Complex ◽  
Loop Mediated Isothermal Amplification

The aim of this study was to develop a simple and reliable method to detect Mycobacterium tuberculosis complex (MTBC) and verify its clinical application preliminarily. A loop-mediated isothermal amplification method coupled with lateral flow biosensor (LAMP-LFB) assay, was developed and evaluated for detection of MTBC. Two sets of primers, which targeted IS6110 and IS1081 sequences of MTBC, were designed for establishment of multiplex LAMP-LFB assay. The amplicons were labelled with biotin and fluorescein isothiocyanate (FITC) by adding FITC labelled primer and biotin-14-dATP and biotin-14-dCTP and could be visualized using LFB. The optimal reaction conditions of multiplex LAMP-LFB assay confirmed were 66°C for 50 min. The analytical sensitivity of multiplex LAMP-LFB is 10 fg of genomic templates using pure culture, and no cross-reactivity with other common bacteria and non-tuberculous mycobacteria strains was obtained. A total of 143 clinical samples collected from 100 TB patients (62 definite TB cases and 38 probable TB cases) and 43 non-TB patients were used for evaluating the feasibility of multiplex LAMP-LFB assay. The multiplex LAMP-LFB (82.0%, 82/100) showed higher sensitivity than culture (47.0%, 47/100, P < 0.001) and Xpert MTB/RIF (54.0%, 54/100, P < 0.001). Importantly, the multiplex LAMP-LFB assay detected additional 28 probable TB cases, which increased the percentage of definite TB cases from 62.0% (62/100) to 90.0% (90/100). The specificity of multiplex LAMP-LFB assay in patients without TB was 97.7% (42/43). Therefore, multiplex LAMP-LFB assay is a simple, reliable, and sensitive method for MTBC detection, especially in probable TB cases and resource limited settings.

Development and application of loop-mediated isothermal amplification label-based nanoparticles lateral flow biosensor for detection of Mycobacterium tuberculosis

2019 ◽  
Author(s):  
Xingyun Wang ◽  
Yi Wang ◽  
Weiwei Jiao ◽  
Guirong Wang ◽  
Yacui Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Diagnostic Technique ◽  
Cost Effective ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
High Morbidity ◽  
Analytical Sensitivity ◽  
Loop Mediated Isothermal Amplification

Abstract Tuberculosis is a serious disease with high morbidity and mortality, thus rapid and cost-effective diagnostic test for Mycobacterium tuberculosis (MTB) is urgently needed. Here, a novel detection diagnostic technique, termed as loop-mediated isothermal amplification label-based nanoparticles with lateral flow biosensor (LAMP-LFB), was developed and evaluated for rapid, reliable and objective detection of MTB. Two sets of primers, which targeted IS 6110 and IS 1081 sequences of MTB, were simultaneously designed for establishment of LAMP-LFB assay. The optimal reaction conditions of MTB-LAMP-LFB assay confirmed were 66ºC for only 50min. The analytical sensitivity of MTB-LAMP-LFB is 10fg of genomic templates in pure culture, and the detection results obtained from LFB was in conformity with agarose gel electrophoresis. No cross-reactivity with other common bacteria and non-tuberculous mycobacteria strains (NTM) was obtained. A total of 158 clinical samples were collected from presumptive 158 TB patients, were used for evaluating the feasibility of MTB-LAMP-LFB assay. Among 98 TB patients diagnosed with composite reference standard, the positive rate for MTB detection using liquid culture, Xpert MTB/RIF and LAMP-LFB were 40.0% (39/98), 50.0% (48/98), and 86.7% (85/98), respectively. Among 39 culture confirmed samples, 84.6% (33/39) cases were Xpert MTB/RIF-positive and 92.3% (36/39) were LAMP-LFB-positive. For the 59 clinically diagnosed TB cases 25.4% (15/59) and 83.0% (49/59) were Xpert MTB/RIF-positive and LAMP-LFB positive, respectively. Therefore, MTB-LAMP-LFB assay is a simple, reliable, and sensitive method for MTB detection and maybe prospective in early diagnosis of MTB.

scholarly journals Development and evaluation of a multiplex loop-mediated isothermal amplification (LAMP) assay for differentiation of Mycobacterium tuberculosis and non-tuberculosis mycobacterium in clinical samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0244753
Author(s):  
Jeeyong Kim ◽  
Borae G. Park ◽  
Da Hye Lim ◽  
Woong Sik Jang ◽  
Jeonghun Nam ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Positive Reaction ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Published Data ◽  
Analytical Sensitivity ◽  
Loop Mediated Isothermal Amplification

Introduction The rapid and accurate diagnosis of tuberculosis (TB) is important to reduce morbidity and mortality rates and risk of transmission. Therefore, molecular detection methods such as a real-time PCR–based assay for Mycobacterium tuberculosis (MTB) have been commonly used for diagnosis of TB. Loop-mediated isothermal amplification (LAMP) assay was believed to be a simple, quick, and cost-effective isothermal nucleic acid amplification diagnostic test for infectious diseases. In this study, we designed an in-house multiplex LAMP assay for the differential detection of MTB and non-tuberculosis mycobacterium (NTM), and evaluated the assay using clinical samples. Material and methods For the multiplex LAMP assay, two sets of specific primers were designed: the first one was specific for IS6110 genes of MTB, and the second one was universal for rpoB genes of mycobacterium species including NTM. MTB was confirmed with a positive reaction with both primer sets, and NTM was identified with a positive reaction by only the second primer set without a MTB-specific reaction. Total 333 clinical samples were analyzed to evaluate the multiplex LAMP assay. Clinical samples were composed of 195 positive samples (72 MTB and 123NTM) and 138 negative samples. All samples were confirmed positivity or negativity by real-time PCR for MTB and NTM. Analytical sensitivity and specificity were evaluated for the multiplex LAMP assay in comparison with acid fast bacilli staining and the culture method. Results Of 123 NTM samples, 121 were identified as NTM and 72/72 MTB were identified as MTB by the multiplex LAMP assay. False negative reactions were seen only in two NTM positive samples with co-infection of Candida spp. All 138 negative samples were identified as negative for MTB and NTM. Analytical sensitivity of the multiplex LAMP assay was 100% (72/72) for MTB, and 98.4% (121/123) for NTM. And the specificity of assay was 100% (138/138) for all. Conclusions Our newly designed multiplex LAMP assay for MTB and NTM showed relatively good sensitivity in comparison with previously published data to detect isolated MTB. This multiplex LAMP assay is expected to become a useful tool for detecting and differentiating MTB from NTM rapidly at an acceptable sensitivity.

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