Sensitive detection of the IS<i>6110</i> sequence of <i>Mycobacterium tuberculosis</i> complex based on PCR-magnetic bead ELISA

Kyaw, Soe Paing; Hanthamrongwit, Jariya; Jangpatarapongsa, Kulachart; Khaenam, Prasong; Leepiyasakulchai, Chaniya

doi:10.1039/c8ra06599c

Cited by 10 publications

(3 citation statements)

References 28 publications

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“…Several companies now sell commercial versions (MagPix TM ) of systems that utilize these advantages, and the immunoassays perform at least as well, or even better, than the conventional ELISA systems [ 148 , 149 ]. The simplified handling of magnetic particles is also of great value in the preparation of samples for quantitative polymerase chain reaction (qPCR), as was demonstrated in the sensitive detection of Tuberculosis pathogens using a magnetic bead to gather sample DNA (e.g., amplicons) [ 150 ].…”

Section: Diagnosticsmentioning

confidence: 99%

Magnetic Nanoparticles in Biology and Medicine: Past, Present, and Future Trends

et al. 2021

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The use of magnetism in medicine has changed dramatically since its first application by the ancient Greeks in 624 BC. Now, by leveraging magnetic nanoparticles, investigators have developed a range of modern applications that use external magnetic fields to manipulate biological systems. Drug delivery systems that incorporate these particles can target therapeutics to specific tissues without the need for biological or chemical cues. Once precisely located within an organism, magnetic nanoparticles can be heated by oscillating magnetic fields, which results in localized inductive heating that can be used for thermal ablation or more subtle cellular manipulation. Biological imaging can also be improved using magnetic nanoparticles as contrast agents; several types of iron oxide nanoparticles are US Food and Drug Administration (FDA)-approved for use in magnetic resonance imaging (MRI) as contrast agents that can improve image resolution and information content. New imaging modalities, such as magnetic particle imaging (MPI), directly detect magnetic nanoparticles within organisms, allowing for background-free imaging of magnetic particle transport and collection. “Lab-on-a-chip” technology benefits from the increased control that magnetic nanoparticles provide over separation, leading to improved cellular separation. Magnetic separation is also becoming important in next-generation immunoassays, in which particles are used to both increase sensitivity and enable multiple analyte detection. More recently, the ability to manipulate material motion with external fields has been applied in magnetically actuated soft robotics that are designed for biomedical interventions. In this review article, the origins of these various areas are introduced, followed by a discussion of current clinical applications, as well as emerging trends in the study and application of these materials.

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Section: Diagnosticsmentioning

confidence: 99%

Magnetic Nanoparticles in Biology and Medicine: Past, Present, and Future Trends

et al. 2021

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“…Even though the IS 6110 -specific PCR assay showed an advantage for its improved sensitivity, it could not differentiate between live and dead mycobacteria, and therefore, it could only be used for screening samples but not for the surveillance of patients in treatment ( Kyaw et al 2018 ; Ambreen et al 2019 ). For this reason, it was imperative to evaluate the accuracy of results obtained by the specific PCR method in the current study using direct culturing on Lowenstein-Jensen media followed by Ziehl-Neelsen staining, which is considered the gold standard in cases of discordant results ( Diriba et al 2017 ).…”

Section: Discussionmentioning

confidence: 99%

Evaluating the Sensitivity of Different Molecular Techniques for Detecting Mycobacterium tuberculosis Complex in Patients with Pulmonary Infection

Hemeg,

Albulushi,

Ozbak

et al. 2023

Polish Journal of Microbiology

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This study aimed to evaluate the accuracy of detecting drug-resistant Mycobacterium tuberculosis complex (MTBC)-specific DNA in sputum specimens from 48 patients diagnosed with pulmonary tuberculosis. The presence of MTBC DNA in the specimens was validated using the GeneXpert MTB/RIF system and compared with a specific PCR assay targeting the IS6110 and the mtp40 gene sequence fragments. Additionally, the results obtained by multiplex PCR assays to detect the most frequently encountered rifampin, isoniazid, and ethambutol resistance-conferring mutations were matched with those obtained by GeneXpert and phenotypic culture-based drug susceptibility tests. Of the 48 sputum samples, 25 were positive for MTBC using the GeneXpert MTB/RIF test. Nevertheless, the IS6110 and mtp40 single-step PCR revealed the IS6110 in 27 of the 48 sputum samples, while the mtp40 gene fragment was found in only 17 of them. Furthermore, multiplex PCR assays detected drug-resistant conferring mutations in 21 (77.8%) of the 27 samples with confirmed MTBC DNA, 10 of which contained single drug-resistant conferring mutations towards ethambutol and two towards rifampin, and the remaining nine contained double-resistant mutations for ethambutol and rifampin. In contrast, only five sputum specimens (18.5%) contained drug-resistant MTBC isolates, and two contained mono-drug-resistant MTBC species toward ethambutol and rifampin, respectively, and the remaining three were designated as multi-drug resistant toward both drugs using GeneXpert and phenotypic culture-based drug susceptibility tests. Such discrepancies in the results emphasize the need to develop novel molecular tests that associate with phenotypic non-DNA-based assays to improve the detection of drug-resistant isolates in clinical specimens in future studies.

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“…By knowing the results, we have improved our decontamination process, restricted access during the staining process, and personal protective equipment to ensure biosafety. Known for its higher sensitivity in M. tuberculosis detection, 13,18,19 qPCR of insertion sequence IS6110 was also performed in this experiment. Lower cycle threshold (Ct) values, indicating a higher load of M. tuberculosis DNA, were detected in areas that include processing of clinical samples and positive culture, such as ZN staining basin, light microscope, incubators, biosafety cabinets, and technician's gloves during the sputum culture process.…”

Section: Discussionmentioning

confidence: 99%

Application of Replicate Organism Detection and Counting Method (RODAC) in Measuring Mycobacterium Tuberculosis Contamination in High Burden Laboratories

Chaidir

Susilawati

Ajie

et al. 2022

AMJ

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Background: Technicians working in high burden tuberculosis (TB) laboratories pose a higher risk of being infected by Mycobacterium tuberculosis from clinical samples. Contamination control is mandatory to detect the release of bacteria into the working environment and to minimize the risk of exposure to the workers. The contamination measurement is rarely performed due to the lack of standard methodology. This study optimized and applied a unique culture-based method named Replicate Organism Detection and Counting (RODAC) plates to assess the presence of M. tuberculosis contaminant in the laboratory with high burden of clinical samples. Methods: RODAC was applied on twenty working surfaces in the Mycobacteriology Laboratory of Universitas Padjadjaran. The results of RODAC were compared with DNA-based detection from the same working surfaces using in-house IS6110 real-time PCR (IS6110-qPCR). The detection limit of the RODAC plate was 19.6 CFU mL-1.Results: From all working surfaces tested, two distinct colonies were found on RODAC plate stamped on the Ziehl-Neelsen staining basin. Those colonies were identified as M. tuberculosis and non-tuberculous mycobacteria (NTM), as confirmed by the MPT64 antigen test and the presence of acid-fast bacilli. IS6110-qPCR detected the presence of M. tuberculosis DNA in ten sampling points, including the ZN staining basin, incubators, and microscopy areas. IS6110-qPCR detected more working surface contamination versus RODAC. However, it was noted that RODAC, which was a culture-based method, detected live bacteria, while PCR could not distinguish between live and dead bacteria.Conclusion: The application of the RODAC plate is more suitable for monitoring the contamination of live bacteria in the working environment and to inform a proper corrective action.

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Sensitive detection of the IS6110 sequence of Mycobacterium tuberculosis complex based on PCR-magnetic bead ELISA

Abstract: Tuberculosis (TB) is ranked as the top killer among infectious diseases worldwide.

Cited by 10 publications

References 28 publications

Magnetic Nanoparticles in Biology and Medicine: Past, Present, and Future Trends

Magnetic Nanoparticles in Biology and Medicine: Past, Present, and Future Trends

Evaluating the Sensitivity of Different Molecular Techniques for Detecting Mycobacterium tuberculosis Complex in Patients with Pulmonary Infection

Application of Replicate Organism Detection and Counting Method (RODAC) in Measuring Mycobacterium Tuberculosis Contamination in High Burden Laboratories

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