Krishna Himmatbhai Goyani

Shri Jagdishprasad Jhabarmal Tibrewala University

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Co-Authors

Pratap N. Mukhopadhyaya

Shri Jagdishprasad Jhabarmal Tibrewala University

Shalin Vaniawala

N-Gene (Hungary)

Dinshaw J. Patel

Memorial Sloan Kettering Cancer Center

Chirajyoti Deb

Arnold Palmer Hospital for Children

Arpan Acharya

University of Nebraska Medical Center

Bakhtiyar Alam Syed

Hemchandracharya North Gujarat University

Harsh Parekh

Wayne State University

Cooperative Institutions

Bose Institute

Indian Institute of Management Calcutta

GenePath Dx (India)

Academy of Scientific and Innovative Research

Memorial Sloan Kettering Cancer Center

Kettering University

Southern University of Science and Technology

Mayo Clinic in Florida

Cornell University

Nanyang Technological University

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Progress in polymerase chain reaction technology and its contribution to the tuberculosis diagnostic ecosystem in India: A mini review

World Journal of Advanced Research and Reviews (2023)

Krishna Himmatbhai Goyani Pratap N. Mukhopadhyaya

The advent of Polymerase Chain Reaction (PCR) has revolutionized the human molecular diagnostic scenario. Its ability to exponentially generate desired stretch of DNA from chromosomal or complimentary DNA has laid the foundation for sensitive and specific detection of a pathogen or an aberrant stretch of DNA. Importantly, PCR technology has helped generate diagnostic immunity from phenotypic variation of pathogenic targets. The technological evolution of conventional end-point PCR to its real time version further added clinically important features such as enhanced sensitivity and quantification of target DNA molecules. Two important chemistries became prominent in the real-time PCR domain. The intercalating dye-based chemistry comes with convenience, economy and feature such as melt curve analysis but also carries the disadvantage of overestimation of signal due to unwanted signal from primer dimers. Dual labelled probe on the other hand provides unprecedented sensitivity and specificity. Isothermal method of genome nucleic acid amplification has also evolved as a parallel and convenient method of amplification-based detection of nucleic acid targets. Tuberculosis diagnostics has immensely benefitted from these technologies. However, unmet need exists in sputum handling methods and in its adaptation for molecular diagnostics in resource limited settings. Significant dependence for diagnostic needs on the western world is a matter of concern in India. The country will benefit from rapid and indigenous research and development of molecular diagnostic solutions by way of formulating kits for rapid extraction of DNA and RNA from various clinical sources and developing detection technologies that can compete international products in its category. A solution to address sputum transportation, storage and DNA extraction that has enhanced advantages compared to current methods will also be helpful in the efforts to make India free from tuberculosis.

Primer (cosmetics)

Molecular diagnostics

Primer dimer

Multiple displacement amplification

10.30574/wjarr.2023.20.3.2658

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Mutations within the Open Reading Frame (ORF) including Ochre stop codon of the Surface Glycoprotein gene of SARS-CoV-2 virus erase potential seed location motifs of human non-coding microRNAs

World Journal of Advanced Research and Reviews (2021)

Krishna Himmatbhai Goyani Shalin Vaniawala Pratap N. Mukhopadhyaya

MicroRNA are short and non-coding RNA, 18-25 nucleotides in length. They are produced at the early stage of viral infection. The roles played by cellular miRNAs and miRNA-mediated gene-silencing in the COVID-19 epidemic period is critical in order to develop novel therapeutics. We analyzed SARS-CoV-2 Surface Glycoprotein (S) nucleotide sequence originating from India as well as Iran, Australia, Germany, Italy, Russia, China, Japan and Turkey and identified mutation in potential seed location of several human miRNA. Seventy single nucleotide polymorphisms (SNP) were detected in the S gene out of which, 36, 32 and 2 were cases of transitions, transversions and deletions respectively. Eleven human miRNA targets were identified on the reference S gene sequence with a score >80 in the miRDB database. Mutation A845S erased a common binding site of 7 human miRNA (miR-195-5p, miR-16-5p, miR-15b-5p, miR-15a-5p, miR-497-5p, miR-424-5p and miR-6838-5p). A synonymous mutation altered the wild type Ochre stop codon within the S gene sequence (Italy) to Opal thereby changing the seed sequence of miR-511-3p. Similar (synonymous) mutations were detected at amino acid position 659 and 1116 of the S gene where amino acids serine and threonine were retained, abolishing potential seed location for miR-219a-1-3p and miR-20b-3p respectively. The significance of this finding in reference to the strategy to use synthetic miRNA combinations as a novel therapeutic tool is discussed.

Coding region

Silent mutation

Stop codon

10.30574/wjarr.2021.10.3.0288

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Evolution of nucleic acid extraction methods and its role in development of complex human diagnostic solutions with special reference to tuberculosis: A review

Magna Scientia Advanced Research and Reviews (2023)

Krishna Himmatbhai Goyani Pratap N. Mukhopadhyaya

Nucleic acid extraction from biological sources has evolved with time and is currently one of the most important contributors to the development of modern human molecular diagnostics. Pure and sufficient quantity of DNA and RNA from limited quantity of cells, which may be fresh or archived, holds key to successful detection of source and cause of many diseases. The challenges of cell disruption to release nucleic acids and the wide range of strategies applied to purify them by removing various enzyme inhibitors are discussed. The demographic spread of the disease of tuberculosis, hurdles faced during transportation of the clinical samples for nucleic acid extraction of Mycobacterium tuberculosis pathogen and different detection technologies supported by the extracted pathogen nucleic acid are reviewed.

Nucleic acid detection

10.30574/msarr.2023.9.2.0166

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Citations (1)

Mutations within the Open Reading Frame (ORF) including Ochre stop codon of the Surface Glycoprotein gene of SARS-CoV-2 virus erase potential seed location motifs of human non-coding microRNAs

bioRxiv (Cold Spring Harbor Laboratory) (2021)

Krishna Himmatbhai Goyani Shalin Vaniawala Pratap N. Mukhopadhyaya

Abstract MicroRNA are short and non-coding RNA, 18-25 nucleotides in length. They are produced at the early stage of viral infection. The roles played by cellular miRNAs and miRNA-mediated gene-silencing in the COVID-19 epidemic period is critical in order to develop novel therapeutics. We analysed SARS-CoV-2 Surface Glycoprotein (S) nucleotide sequence originating from India as well as Iran, Australia, Germany, Italy, Russia, China, Japan and Turkey and identified mutation in potential seed location of several human miRNA. Seventy single nucleotide polymorphisms (SNP) were detected in the S gene out of which, 36, 32 and 2 were cases of transitions, transversions and deletions respectively. Eleven human miRNA targets were identified on the reference S gene sequence with a score >80 in the miRDB database. Mutation A845S erased a common binding site of 7 human miRNA (miR-195-5p, miR-16-5p, miR-15b-5p, miR-15a-5p, miR-497-5p, miR-424-5p and miR-6838-5p). A synonymous mutation altered the wild type Ochre stop codon within the S gene sequence (Italy) to Opal thereby changing the seed sequence of miR-511-3p. Similar (synonymous) mutations were detected at amino acid position 659 and 1116 of the S gene where amino acids serine and threonine were retained, abolishing potential seed location for miR-219a-1-3p and miR-20b-3p respectively. The significance of this finding in reference to the strategy to use synthetic miRNA combinations as a novel therapeutic tool is discussed.

Coding region

Silent mutation

Stop codon

10.1101/2021.06.19.449095

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Mutations within the Open Reading Frame (ORF) including Ochre stop codon of the Surface Glycoprotein gene of SARS-CoV-2 virus erase potential seed location motifs of human non-coding microRNAs

Zenodo (CERN European Organization for Nuclear Research) (2021)

Krishna Himmatbhai Goyani Shalin Vaniawala Pratap N. Mukhopadhyaya

Stop codon

Coding region

10.5281/zenodo.5075302

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Sputum-spotted solid matrix designed to release diagnostic-gradeMycobacterium tuberculosisDNA demonstrate optimal biocontainment property

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Krishna Himmatbhai Goyani Pratap N. Mukhopadhyaya

Abstract Biosafety quotient of a reagent-coated cellulose matrix designed to release Mycobacterium tuberculosis (Mtb) DNA from sputum deposited onto it, was determined. Thirty-seven sputum samples infected with Mtb and 5 sputum samples from healthy individuals were processed and spotted onto the TBSend cards. Live Mtb bacilli were attempted for rescue from the spotted TBSend cards by washing them in phosphate buffer under mild shaking conditions. No live Mtb bacilli could be detected by the BACTEC™ MGIT 960™ TB System from both Mtb infected as well as non-infected, sample-spotted TBSend cards when they were washed at two different time-points, viz ., 1 minute and 15 hours from the time of spotting of the cards with sputum mixed with the preprocessing “spotting” buffer. The study reiterated published finding that chaotropic agents, which is an active component of the TBSend card module, have cent per cent bactericidal properties with regard to Mtb .

Bedaquiline

10.1101/2023.08.25.554768

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Sputum-Spotted Solid Matrix Designed to Release Diagnostic-Grade Mycobacterium tuberculosis DNA Demonstrate Optimal Biocontainment Property

International Journal of Science and Research (IJSR) (2023)

Krishna Himmatbhai Goyani Pratap N. Mukhopadhyaya

Background: Biosafety quotient of a reagent-coated cellulose matrix designed to release Mycobacterium tuberculosis (Mtb) DNA from sputum deposited onto it was determined. Method: Thirty-seven sputum samples infected with Mtb and 5 sputum samples from healthy individuals were processed and spotted onto a chemically coated cellulose matrix named the TBSend cards. Live Mtb bacilli were attempted for rescue from the spotted TBSend cards by washing them in phosphate buffer saline under mild shaking conditions. No live Mtb bacilli could be detected by the BACTEC™ MGIT 960™ TB System from both Mtb infected and Mtb noninfected sample-spotted TBSend cards when they were washed at two different time-points namely, 1 minute and 15 hours from the time of spotting of the cards with processed sputum. Result: The study consolidated published findings that chaotropic agents, which is an active component of the TBSend card module, have cent per cent bactericidal properties with regard to Mtb. Conclusion: Mtb infected sputum mixed with a biological cell-lysing solution and spotted onto a cellulose matrix coated with chaotropic salts effectively eliminates all live pathogen and can be used as a bio-safe Tb sputum transportation, storage and DNA-release device for Mtb NAAT.

Matrix (chemical analysis)

10.21275/sr231013144015

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Citations (4)

Estimating limit of detection of M.tuberculosisDNA as an analyte released from a chemically coated solid matrix and using a WHO-approved CB-NAAT platform

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Krishna Himmatbhai Goyani Pratap N. Mukhopadhyaya

Abstract The limit of detection of Mtb ( Mycobacterium tuberculosis ) bacilli was evaluated by detection of released DNA from a chemically treated cellulose matrix. Loopful of the Mtb cells were suspended in phosphate buffer (pH 6.8), DNA extracted from it and quantified using a commercial Mtb real time PCR quantitation kit. Synthetic sputum, prepared using acrylamides as the matrix of choice was mixed with defined number of Mtb bacilli and spotted onto the cellulose matrix. Mtb DNA from the matrix was released into a DNA-release buffer and detected using a WHO-approved CB-NAAT detection platform. One hundred and fifty, 500 and 1000 but not 10 Mtb bacilli/mL of synthetic sputum could be detected by this protocol. The result underlined the potential of cellulose matrix as a potent, room temperature, Mtb-infected sputum transportation, storage and DNA-release device for sensitive detection of Mtb using Mtb Xpert Ultra CB-NAAT as the WHO-approved Mtb test of choice.

Matrix (chemical analysis)

10.1101/2023.09.26.558062

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Implications of Molecular Estimation of Allelic Burden of JAK2 V617F Mutant Allele in Essential Thrombocythemia and Polycythemia Vera Diseases

Bakhtiyar Alam Syed Krishna Himmatbhai Goyani Shalin Vaniawala Harsh Parekh Arpan Acharya

JAK2 V617F

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Enhancement of M. tuberculosis Line Probe Assay Sensitivity through Whole Genome Amplification of Low-Quantity DNA Released from Sputum and Archived on Chemically-Coated Cellulose Matrix Using an Isothermal Enzymatic Strand-Displacement Process

medRxiv (Cold Spring Harbor Laboratory) (2024)

Krishna Himmatbhai Goyani Chirajyoti Deb Dinshaw J. Patel Shalin Vaniawala Pratap N. Mukhopadhyaya

Abstract In this study, thirty-nine sputum samples from tuberculosis (TB)-positive patients undergoing first-line therapy were collected and archived on a chemical-coated cellulose matrix. DNA was extracted from these matrices and tested for Mycobacterium tuberculosis using the Xpert MTB/RIF Ultra assay. Seven samples tested positive for M. tuberculosis , with low levels of detection. End-point PCR yielded faint signals in four samples, but no signal in the remaining three. A Line Probe Assay (LPA) detected pathogen DNA in only one of the three PCR-negative samples. Remarkably, the LPA-negative samples were successfully detected by LPA and end-point PCR following isothermal, strand displacement-based whole genome amplification (WGA) of the stock DNA. The drug sensitivity profile of these samples was consistent with the Xpert MTB/RIF Ultra results obtained from the original stock DNA. Additionally, sputum DNA from healthy individuals spiked with 125 ng, but not with 1.25 ng, of M. tuberculosis culture DNA was detected by LPA. Following WGA, the 1.25 ng sample was also detected successfully by LPA, and its drug sensitivity profile matched that of the 125-ng sample. These findings indicate that WGA of sputum DNA from a cellulose matrix, even with low pathogen load, can enhance the detection capabilities of LPA by enriching the genome target through an isothermal enzymatic strand displacement method. This method holds promise for improving diagnostic sensitivity in TB detection.

Multiple displacement amplification

10.1101/2024.06.27.24309624

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