Fig 1.
Schematic representation of the experimental workflow.
Patients attending the Department of Thisiology and Leprosy of the Universidade Luterana do Brasil (ULBRA) were included in the study if had not had previous TB treatment and were able to produce a minimum of 500 μL of sputum per collection day. Accordingly, patients were excluded if had previous TB treatment or were unable to produce the minimal sample volume. Samples were collected on two consecutive days and were subjected to the GeneXpert MTB/Rif assay and culture. Next, eight liquefying solutions and six elution protocols were evaluated using the same samples. Eluted DNA were evaluated by two qPCR instruments and two reagents’ storage formats.
Table 1.
Liquefaction of mucin with different chemicals.
Table 2.
Steps of each of the six different DNA elution protocols evaluated in the present work.
Table 3.
Sequence of primers and probes used for detection of the genomic MTB target IS6110 and of the human gene 18S rRNA.
Fig 2.
Representative traces of the qPCR amplification of M. tuberculosis IS6110 target.
Porcine mucin samples spiked with M. tuberculosis H37Rv DNA (25 ng/μL) were processed according to protocols #3 or #4. Traces “a” and “b” were obtained after 1:10 and 1:100 dilution of the eluted DNA obtained with protocol #3. Traces “c” and “d” were obtained after 1:10 and 1:100 dilution of the eluted DNA obtained with protocol #4. “PC” and “NC” represent the positive control (25 ng/μL of DNA extracted from H37Rv MTB cells) and the negative control (TE pH 8.0), respectively. Traces “e”, “f”, “g”, and “h”, (“e–h”) represent 1:100 dilutions of protocols #1, #2, #5, and #6. Traces are representative of at least three independent extraction procedures.
Table 4.
Comparison of ABI7500 and Q3 using protocols #3 and #4.
Table 5.
Colony forming unit and IS6110 detection by two qPCR instruments using 1:10 dilutions of a M. tuberculosis cell suspension.
Table 6.
Comparison of detection of M. tuberculosis among different platforms.
Fig 3.
Comparative flowchart of the proposed simplified protocol.
On the left panel, a general procedure for spin-column DNA extraction used in most commercial products. On the right panel, a step-by-step description of the simple and straightforward protocol for POC screening of tuberculosis-suspected samples.