Comparison of DNA Extraction Protocols and Molecular Targets to Diagnose Tuberculous Meningitis

Palomo, Flavia Silva; Rivero, Martha Gabriela Celle; Quiles, Marcos G.; Pinto, Fernando Pereira; Machado, Antônia Maria de Oliveira; Pignatari, Antônio Carlos Campos

doi:10.1155/2017/5089046

“…The disadvantage of PCR is its high cost, molecular lab infrastructure and skilled technologist. At present-day, in India real-time PCR is suitable as a public tool for rapid and early detection of TB [21]. Nanotech assisted PCR with higher efficiencies has been evolved [22][23][24].…”

Section: Discussionmentioning

confidence: 99%

“…These results are found superior than conventional methods. The magnetic nanoparticle (MNP) assisted DNA extraction protocol showed better results in terms of quantification and sensitivity of PCR diagnosis of Mycobacterium tuberculosis, evolving nanotech assisted innovative method [12].…”

Section: Introductionmentioning

confidence: 99%

Evolution of Nanotech Assisted PCR Diagnosis of Mycobacterium Tuberculosis and Its Assessment With Conventional Methods

Sawant

¹

,

Desai

²

,

Patil

³

et al. 2018

Int J Pharm Pharm Sci

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Objective: This study was focused on assessment and performance of conventional and nanotech assisted methods for detection of Mycobacterium tuberculosis. Tuberculosis (TB) still leftovers one of the top ten causes of death worldwide, thus this study has undertaken the use of MNP for early detection of TB.Methods: A cross-sectional studies were conducted on clinical and radiological suspected TB patients in the department of microbiology at D. Y. Patil hospital Kolhapur. All samples received in the month of August 2016 to January 2017. Total One hundred twenty-sputum samples were processed for diagnosis of TB by ZN stain, culture on L. J. Medium and real-timePCR tests.Results: In our present study, 60.83% (73) patient male and 39.17 % (47) were female patient, showing a borderline male prevalence found in our study. Eighteen percent patients were found to be in the age group (21-30 y) are mostly affected for pulmonary tuberculosis. A significant difference was seen in the percentage of differentDNA extraction methods, the fig. being conventional chloroform-phenol 66.66%, the commercial kit 80%, magnetic bead 86.66% and MNPs method 99.66% found to be significant (P<0.0001**).Conclusion: In the present study the MNP-DNA extraction techniques with NALC followed by IS6110 target amplification were found superior for diagnosis of TB. The MNP assisted extraction method showed better results in terms of quantification and sensitivity of TB PCR diagnosis, evolving nanotech assisted innovative method.

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“…Another study by Tang et al reports an integrated device employing HDA for detection of S. typhimurium in various spiked samples 23 . The residual lysis and the precipitation buffer components used in these studies are likely to have reduced the sensitivity of nucleic acid amplification 24,25 . While there are various reports of integrated assays on paper (Table 1), due to extensive washing steps to filter amplification inhibitors there is an increase in the total assay time and complexity, which limits the translation of these assays.…”

Section: Introductionmentioning

confidence: 99%

An integrated one-step assay combining thermal lysis and loop-mediated isothermal DNA amplification (LAMP) in 30 min from E. coli and M. smegmatis cells on a paper substrate

Naik

¹

,

Jaitpal

²

,

Shetty

³

et al. 2019

Sensors and Actuators B: Chemical

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Developing sensors in the domains of food safety, soil analysis, water quality monitoring and healthcare often requires distinguishing between different species of bacteria. The most rapid, sensitive and specific method to identify bacteria is by analysing their DNA sequence, which comprises of disinfection and lysis of bacterial cells, amplification of the isolated DNA and detection of the amplified sequence. Seamless integration of these assays on a paper substrate remains a big challenge in paperfluidic nucleic acid analyis.Combining lysis and isothermal amplification in a single reaction step is difficult because the porosity of paper and the presence of cell debris following lysis reduces the efficiency of DNA amplification. On the other hand, extracting and purifying the DNA after lysis to improve the amplification efficiency involves addition of chemical reagents, one or more wash steps and manual intervention. This problem is even more complex for mycobacteria as its thick cell wall structure impedes lysis and the high GC-content of the genome requires careful optimization of enzymatic denaturation during isothermal amplification. Here we successfully combine thermal lysis and loop-mediated isothermal amplification (LAMP) into a single reaction step on paper without the need for any intermediate intervention. We demonstrate our integrated assay by amplifying DNA from 100 CFU/mL of Escherichia coli (MG1655) and Mycobacterium smegmatis (mc 2 155) cells in 30 min on a paper substrate. We also confirm that E. coli and M. smegmatis can be completely disinfected on paper by heating at 60 o C for 5 min and 15 min respectively, making this assay safe and suitable for incorporation into diverse paperfluidic sensors for field use.

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“…Secondly, the background signal from lysates should be much lower compared to the signal 23 . The residual lysis and the precipitation buffer components used in these studies are likely to have reduced the sensitivity of nucleic acid amplification 24,25 . While there are various reports of integrated assays on paper (Table 1), due to extensive washing steps to filter amplification inhibitors there is an increase in the total assay time and complexity, which limits the translation of these assays.…”

Section: Introductionmentioning

confidence: 99%

An integrated one-step assay combining thermal lysis and loop-mediated isothermal DNA amplification (LAMP) in 30 min fromE. coliandM. smegmatiscells on a paper substrate

Naik

¹

,

Jaitpal

²

,

Shetty

³

et al. 2019

Preprint

View full text Add to dashboard Cite

Developing sensors in the domains of food safety, soil analysis, water quality monitoring and healthcare often requires distinguishing between different species of bacteria. The most rapid, sensitive and specific method to identify bacteria is by analysing their DNA sequence, which comprises of disinfection and lysis of bacterial cells, amplification of the isolated DNA and detection of the amplified sequence. Seamless integration of these assays on a paper substrate remains a big challenge in paperfluidic nucleic acid analyis.Combining lysis and isothermal amplification in a single reaction step is difficult because the porosity of paper and the presence of cell debris following lysis reduces the efficiency of DNA amplification. On the other hand, extracting and purifying the DNA after lysis to improve the amplification efficiency involves addition of chemical reagents, one or more wash steps and manual intervention. This problem is even more complex for mycobacteria as its thick cell wall structure impedes lysis and the high GC-content of the genome requires careful optimization of enzymatic denaturation during isothermal amplification. Here we successfully combine thermal lysis and loop-mediated isothermal amplification (LAMP) into a single reaction step on paper without the need for any intermediate intervention. We demonstrate our integrated assay by amplifying DNA from 100 CFU/mL of Escherichia coli (MG1655) and Mycobacterium smegmatis (mc 2 155) cells in 30 min on a paper substrate. We also confirm that E. coli and M. smegmatis can be completely disinfected on paper by heating at 60 o C for 5 min and 15 min respectively, making this assay safe and suitable for incorporation into diverse paperfluidic sensors for field use.

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Comparison of DNA Extraction Protocols and Molecular Targets to Diagnose Tuberculous Meningitis

Cited by 9 publications

References 29 publications

Evolution of Nanotech Assisted PCR Diagnosis of Mycobacterium Tuberculosis and Its Assessment With Conventional Methods

Evolution of Nanotech Assisted PCR Diagnosis of Mycobacterium Tuberculosis and Its Assessment With Conventional Methods

An integrated one-step assay combining thermal lysis and loop-mediated isothermal DNA amplification (LAMP) in 30 min from E. coli and M. smegmatis cells on a paper substrate

An integrated one-step assay combining thermal lysis and loop-mediated isothermal DNA amplification (LAMP) in 30 min fromE. coliandM. smegmatiscells on a paper substrate

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