Abstract
We sought to develop a smooth and low cost sample preparation and DNA extraction protocol, streamlined with a ready-to-use qPCR in a portable instrument to overcome some of the existing hurdles. Several solutions were evaluated as to their ability to liquefy a mucin-based matrix. Each liquefied matrix, supplemented with either Mycobacterium tuberculosis (MTB) H37Rv strain DNA or intact cells, was aliquoted onto a filter paper embedded with solubilizing agents, and was subsequently dried up. Most of the nucleic acids, including genomic DNA from the bacilli and the host, binds to the filter paper. Next, several protocols were evaluated to elute the DNA from the paper, using qPCR to detect the insertion sequence IS6110, a M. tuberculosis complex genomic marker. The limit of detection (LOD) of the best protocol was then evaluated using parallel seeding and colony counting. The protocol was also evaluated using seventeen sputum samples, previously characterized by the GeneXpert or culture. Two instruments (the ABI7500 Standard and the Q3-Plus system) and two reagents storage formats (frozen or ready-to-use) were evaluated. Solutions containing guanidine isothiocyanate exerted the best liquefying effect on the mucin-based matrix extracted from one 6-mm punches, followed by a brief incubation at 95°C. The resulting DNA contained impurities, but a simple 1:10 dilution elicited the detection of MTB and human genomic targets. The described protocol presented an apparent LOD of 02 CFU/mL of MTB. Challenging the protocol with previously characterized samples showed substantial agreement with GeneXpert MTB/RIF results (sensitivity of 90%, agreement of 88.9%, kappa coefficient of 0.77), and moderate agreement with culture results (sensitivity of 100%, agreement of 78.9%, kappa coefficient of 0.58). This work presents a sensitive proof-of-concept protocol for sputum liquefaction and decontamination followed by a simple DNA extraction procedure, in which the extraction steps are streamlined with a ready-to-use qPCR in a portable instrument that can be employed in low infrastructure settings.
Highlights
Tuberculosis (TB), caused by the bacillus Mycobacterium tuberculosis (MTB), is one of the leading causes of mortality from a single infectious microorganism, which disproportionately affects low and middle-income countries
We hypothesized that concentrated GSCN and other chemicals would liquefy a sputum-like matrix consisting of porcine mucin
Fast MTB screening for POC settings work, since we propose the sample to be manipulated in low-resource settings, which might not have safety cabinets available for diagnostic routines
Summary
Tuberculosis (TB), caused by the bacillus Mycobacterium tuberculosis (MTB), is one of the leading causes of mortality from a single infectious microorganism, which disproportionately affects low and middle-income countries. In 2016, 1.7 million people have died and 10.4 million been fell ill due to TB, while 56% of the cases were from only five countries: India, Indonesia, China, Philippines and Pakistan [1]. Many diagnostic tests already exist, but a large gap of 4.1 million people without proper TB diagnosis still poses a serious global health challenge [2]. TB diagnosis is routinely performed by sputum culturing, smear microscopy, chest x-rays, the tuberculin skin test, or nucleic acid-based molecular tests [3]. Nucleic acid amplification and detection techniques such as PCR and LAMP have open a new era of specific and sensitive diagnostic testing, these tests are mostly available in developed countries and resource rich areas. Reasons for the lack of widespread use are many, but costs, cumbersome sample preparation and nucleic acid extraction protocols as well as fragile instruments are the most prominent ones [3, 4]
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