Detection of Mycobacterium tuberculosis Complex in Sputum Samples Using Droplet Digital PCR Targeting mpt64

Abstract

Tuberculosis (TB) is one of the top 10 causes of death worldwide. It is challenging to find methods of diagnosis of active pulmonary TB that are sensitive enough to detect cases for proper treatment before unintentional transmission. Droplet digital PCR (ddPCR) is a highly sensitive method to detect genetic material of pathogens, but it has rarely been used for diagnosis of TB. This study compared the sensitivity of ddPCR with that of GeneXpert and AFB smear microscopy in 180 leftover sputum samples from patients suspected of having TB on the basis of clinical symptoms and radiography. Absolute quantification of copy numbers of MTB-specific genes was possible using ddPCR targeting the mpt64 gene. Among the 180 samples, 41.1% were diagnosed as having TB using ddPCR. The sensitivities of AFB smear microscopy, GeneXpert and ddPCR were 41.9%, 82.4% and 100%, respectively. AFB smear microscopy and GeneXpert both had a specificity of 100%, and the specificity of ddPCR was 95.3%. The accuracy of ddPCR (97.2%) is higher than that of GeneXpert (92.7%). This robust ddPCR system could potentially be used as a method for early diagnosis of TB.

Present and Future Applications of Digital PCR in Infectious Diseases Diagnosis

“…A similar study also published recently on 74 tuberculosis patients diagnosed by culture, dPCR, qPCR, and acid-fast staining showed a sensitivity of dPCR of 100%, compared to 82.4% for qPCR and 41.9% for acid-fast bacilli (AFB) smear microscopy [39].…”

Heteroresistance of Mycobacterium tuberculosis in the Sputum Detected by Droplet Digital PCR

“…In our study, 79 MTB-complex detected samples were used and obtained from new TB cases with ddPCRs using the mpt64 assay from a previous study [ 18 ]. The study revealed that the average age of the new TB cases was 53.6 ± 16.0 years.…”

“…In this method, we used 79 MTB-complex detected samples with ddPCRs using mpt64 assay from our previous study [ 18 ]. We sought to detect the heteroresistance associated with mutation in the katG gene at codon 315 and the rpoB gene at codon 531 with ddPCRs using mutation detection assays for these two single-nucleotide polymorphisms (SNPs).…”

“… Concentration = −In (N neg /N)/V droplet where In = inputting n, N neg = number of negative droplets, N = total number of droplets, and Vdroplet = volume of droplets. The calculation of the DNA concentration in the sputum was used in the following formulas [ 18 ]: Total copies = (CPM × V reaction /V used DNA ) × V used DNA × (C total DNA /C used DNA ) where CPM = CPM in the reaction mixture, V reaction = volume of the total reaction, V used DNA = volume of the used DNA, C total DNA = concentration of the total DNA, and C used DNA = concentration of the used DNA. To estimate the CPM in the sputum samples, the following equation was used: CPM in sputum = Total copies in sputum/Volume of sputum …”

Microbial evolution through horizontal gene transfer by mobile genetic elements

“…Each droplet has a picolitre‐to‐nanolitre‐sized volume and is separated by oil; therefore, each PCR reaction is conducted independently. ddPCR can quantify the precise copy number of targeted genes; thus, it is used to detect the targeted bacteria in the microbial community (Aung et al., 2023 ). High‐throughput chromosome conformation capture (Hi‐C) is another method used to identify bacteria that carry a target gene, including an ARG and a plasmid‐specific gene.…”