EasyNAT MTC assay: A simple, rapid, and low-cost cross-priming amplification method for the detection of mycobacterium tuberculosis suitable for point-of-care testing

Abstract

More sensitive, rapid, and affordable diagnostic tools for pulmonary tuberculosis (PTB) are urgently needed. This study aimed to assess the performance of EasyNAT MTC(abbreviation: EasyNAT) (Ustar Biotechnologies, China), a novel isothermal amplification method with a turnaround time of less than two hours that requires a few manual steps to process the sputum.Sputum samples from 249 patients with suspected PTB were subjected to smear, culture, Xpert MTB/RIF (Cepheid, USA) and EasyNAT assay testing. Of the 169 PTB patients, EasyNAT detected more PTB patients than Xpert (72.19% vs. 61.54%, P＜0.05, χ 2 = 4.326). Both the Xpert assay and EasyNAT assay detected almost all the culture-positive sputa successfully, but EasyNAT yielded more positive results among the smear-negative and culture-negative PTB cases (44.59% (33/74) vs. 22.97% (17/74), P＜ 0.01, χ 2 = 7.732). Although the specificity of EasyNAT was lower in contrast to Xpert [95.00% (76/80) vs. 98.75% (79/80)], the difference was not significant (P = 0.363, χ 2 = 0.826). EasyNAT could be used as an initial test for PTB diagnosis due to its simplicity, rapid turnaround time, high sensitivity, and low cost.

A Novel Cross-Priming Amplification-Based Assay for Tuberculosis Diagnosis in Children Using Gastric Aspirate

“…The first generation EasyNAT TB Isothermal Amplification Diagnostic Kit (EasyNAT TB IAD) and the next-generation EasyNAT MTC assay used here have been evaluated in the diagnosis of TB in adults ( Ou et al, 2014 ; Zhang et al, 2021 ). One study reported that the sensitivity of EasyNAT TB IAD in adults with culture-confirmed TB was 84.1% ( Ou et al, 2014 ).…”

“…One study reported that the sensitivity of EasyNAT TB IAD in adults with culture-confirmed TB was 84.1% ( Ou et al, 2014 ). The next-generation EasyNAT MTC assay performed in a clinical setting demonstrated an overall sensitivity of 72.19%, which further increased to 86.84% in cases with confirmed TB using sputum ( Zhang et al, 2021 ). Compared with these findings, the sensitivity of EasyNAT was lower in children.…”

“…Among these, CPA is a powerful, innovative amplification method and has been used in the detection of various pathogens, such as infectious spleen and kidney necrosis virus, Salmonella enterica serovar Indiana, and Staphylococcus aureus ( Qiao et al, 2015 ; Liu et al, 2018 ; Wang et al, 2018 ). A novel kit, the EasyNAT MTC assay (EasyNAT, Ustar, Biotechnologies Co. Ltd., China) based on the CPA technique, has been developed to diagnose TB in adults using sputum and has demonstrated good accuracy ( Zhang et al, 2021 ). In this kit, two cross primers targeting the insertion sequence IS 6110 were designed to specifically detect MTC and improve sensitivity.…”

“…In this kit, two cross primers targeting the insertion sequence IS 6110 were designed to specifically detect MTC and improve sensitivity. The assay is carried out in a sealed tube to prevent cross-contamination and the DNA extraction, DNA purification, amplification, and detection of the target gene are performed in three separate chambers within the cartridge ( Zhang et al, 2021 ). As a quick point-of-care test, the results can be reported within 2 h, which can provide early diagnosis for TB.…”

GeneXpert MTB/RIF combined with conventional methods for tuberculosis in Shanghai Regional Medical Center: a retrospective diagnostic study

“…If disease detection and treatment are delayed, the chance of transmission will increase (3). Therefore, rapid and accurate diagnosis of Mtb is an essential component of treating TB and reducing its global burden (4). Smear microscopy and culture are usually used as routine methods to detect Mtb in laboratories.…”