scholarly journals Ultrasensitive detection of Mycobacterium tuberculosis by a rapid and specific probe-triggered one-step, simultaneous DNA hybridization and isothermal amplification combined with a lateral flow dipstick

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Wansadaj Jaroenram ◽  
Jantana Kampeera ◽  
Narong Arunrut ◽  
Sarawut Sirithammajak ◽  
Sarinya Jaitrong ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Hybridization ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
High Resource ◽  
Lateral Flow Dipstick ◽  
Probe System ◽  
One Step

Abstract Mycobacterium tuberculosis (Mtb) is an insidious scourge that has afflicted millions of people worldwide. Although there are many rapid methods to detect it based on loop-mediated isothermal amplification (LAMP) and a lateral flow dipstick (LFD), this study made further improvements using a new set of primers to enhance LAMP performance and a novel DNA probe system to simplify detection and increase specificity. The new probe system eliminates the post-LAMP hybridization step typically required for LFD assays by allowing co-hybridization and amplification of target DNA in one reaction while preventing self-polymerization that could lead to false-positive results. The improved assay was named Probe-Triggered, One-Step, Simultaneous DNA Hybridization and LAMP Integrated with LFD (SH-LAMP-LFD). SH-LAMP-LFD was simpler to perform and more sensitive than previously reported LAMP-LFD and PCR methods by 100 and 1000 times, respectively. It could detect a single cell of Mtb. The absence of cross-reactivity with 23 non-TB bacteria, and accurate test results with all 104 blind clinical samples have highlighted its accuracy. Its robustness and portability make SH-LAMP-LFD suitable for users in both low and high resource 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 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.

scholarly journals Detection ofMycobacterium tuberculosisby Using Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick in Clinical Samples

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Thongchai Kaewphinit ◽  
Narong Arunrut ◽  
Wansika Kiatpathomchai ◽  
Somchai Santiwatanakul ◽  
Pornpun Jaratsing ◽  
...  
Keyword(s):  
Communicable Disease ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Lamp Method ◽  
Cross Hybridization ◽  
Persistent Problem ◽  
Loop Mediated Isothermal Amplification ◽  
Lateral Flow Dipstick ◽  
Epidemiological Surveys

Tuberculosis (TB) is a communicable disease caused by the bacteriumMycobacterium tuberculosis(MTB) and is a persistent problem in the developing countries. Loop-mediated isothermal amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. Here, a LAMP method was combined with a chromatographic lateral-flow dipstick (LFD) to detect IS6110gene ofM. tuberculosisspecifically and rapidly. The reaction was optimized at 63°C for 60 min, and the amplified DNA hybridized to an FITC-labeled oligonucleotide probe for 5 min was detected at the LFD test line 5 min after application. Excluding the step of DNA extraction, the test results could be generated approximately within 1 h. In addition to the advantage of short assay time, this technique could avoid the contact of carcinogenic ethidium bromide due to the exclusion of the electrophoresis analysis step. Furthermore, the data indicated that LAMP-LFD could detectM. tuberculosisgenomic DNA as little as 5 pg. The technique showed a significant specificity since no cross-hybridization toM. intracellulare(MIC),M. fortuitum(MFT),M. avium(MAV),M. kansasii(MKS), andM. gordonae(MGD) genomic DNAs was observed. In the clinical unknown samples test, the sensitivity of LAMP-LFD was 98.92 % and the specificity was 100 % compared to those of the standard culture assay. Based on its sensitivity, specificity, rapidity, low cost, and convenience, LAMP-LFD could be applicable for use in both laboratories and epidemiological surveys of MTB.

scholarly journals Recombinase-Aided Amplification Coupled with Lateral Flow Dipstick for Efficient and Accurate Detection of Porcine Parvovirus

Life ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 762
Author(s):  
Yihong He ◽  
Wenxian Chen ◽  
Jindai Fan ◽  
Shuangqi Fan ◽  
Hongxing Ding ◽  
...  
Keyword(s):  
Detection Method ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Porcine Parvovirus ◽  
Swine Industry ◽  
Resource Limited ◽  
Efficient Detection ◽  
Lateral Flow Dipstick ◽  
Polymerase Chain ◽  
Embryonic Death

Porcine parvovirus (PPV) infection is the primary cause of SMEDI (stillbirth; mummification; embryonic death; infertility) syndrome, which is a global burden for the swine industry. Thus, it is crucial to establish a rapid and efficient detection method against PPV infection. In the present work, we developed a recombinase-aided amplification (RAA) assay, coupled with a lateral flow dipstick (LFD), to achieve an amplification of PPV DNA at 37 °C within 15 min. The detection limits of PPV RAA-LFD assay were 102 copies/μL recombinant plasmid pMD19-T-VP1, 6.38 × 10−7 ng/μL PPV DNA, and 10−1 TCID50/mL virus, respectively. This method was highly specific for PPV detection with no cross-reactivity for other swine pathogens. In contrast to polymerase chain reaction (PCR), the PPV RAA-LFD assay is more sensitive and cost-saving. Hence, the established PPV RAA-LFD assay provided an alternative for PPV detection, especially in resource-limited regions.

scholarly journals Simple and Feasible Detection of Hepatitis B Virus via Combination of Multienzyme Isothermal Rapid Amplification and Lateral Flow Dipstick Strip

2021 ◽  
Vol 8 ◽  
Author(s):  
Mao-Ling Sun ◽  
Hai-Yun Lai ◽  
Na-Yu Chong ◽  
Dong-Fan Liu ◽  
Zhen-Yi Zhang ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Public Health Problem ◽  
Lateral Flow ◽  
Detection Sensitivity ◽  
Clinical Samples ◽  
Major Public Health Problem ◽  
Lateral Flow Dipstick ◽  
B Virus ◽  
Rapid Amplification

Hepatitis B virus infection is not only a huge burden in the field of social health but also a major public health problem that affects the lives and health of the people. Simple, rapid, feasible detection of HBV is critical for its prevention and spread, especially in the developing countries with low-resource laboratories. To this end, we combined multienzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) strip to detect HBV. A pair of primers targeting the conserved region of HBV genome was designed and used in MIRA-LFD assay. Our results found that the entire amplification of MIRA-LFD only takes 10 min at 37°C and the dilution of the amplification products was added in the LFD strip and observed by the naked eye after 10 min. The detection sensitivity of this method can reach 10 pg. The 45 clinical samples were detected by MIRA-LFD and real-time PCR. The accuracy rate of MIRA-LFD was 100%. Therefore, these characteristics of our newly developed MIRA-LFD assay make it particularly useful and suitable for detecting HBV in the resource-limited condition.

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