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

Wansadaj Jaroenram,Narong Arunrut,Wansika Kiatpathomchai,Sarinya Jaitrong,Sarawut Sirithammajak,Angkana Chaiprasert,Pakapreud Khumwan,Kobporn Boonnak,Jantana Kampeera,Therdsak Prammananan

doi:10.1038/s41598-020-73981-6

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.

Highlights

Mycobacterium tuberculosis (Mtb) is an insidious scourge that has afflicted millions of people worldwide
In an initial attempt to address this problem, we reported in 2013 a method to detect Mtb[12] based on loopmediated isothermal amplification (LAMP)[13] in combination with a lateral flow dipstick (LFD) assay
We evaluated the diagnostic performance of our SH-loop-mediated isothermal amplification (LAMP)-LFD with 104 sputum-derived DNA samples isolated from TB and non-TB patients, 74 test samples were identified as positive for Mtb while the remainders were determined as negative

Summary

Introduction

Mycobacterium tuberculosis (Mtb) is an insidious scourge that has afflicted millions of people worldwide. 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. The method demonstrated high specificity for Mtb detection in a total assay time of approximately 70 min This early LAMP-LFD platform successfully addressed some existing limitations of TB diagnosis, it was open to further improvement to reduce the number of steps required to complete the assay, regarding the hybridization of LAMP amplicons to the LFD probe.

Methods

Results

Conclusion