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Papers by Sirintip Dangtip

Talanta, 2022

Colorimetric loop-mediated DNA isothermal amplification-based assays have gained momentum in the ... more Colorimetric loop-mediated DNA isothermal amplification-based assays have gained momentum in the diagnosis of COVID-19 owing to their unmatched feasibility in low-resource settings. However, the vast majority of them are restricted to proprietary pH-sensitive dyes that limit downstream assay optimization or hinder efficient result interpretation. To address this problem, we developed a novel dual colorimetric RT-LAMP assay using in-house pH-dependent indicators to maximize the visual detection and assay simplicity, and further integrated it with the artificial intelligence (AI) operated tool (RT-LAMP-DETR) to enable a more precise and rapid result analysis in large scale testing. The dual assay leverages xylenol orange (XO) and a newly formulated lavender green (LG) dye for distinctive colorimetric readouts, which enhance the test accuracy when performed and analyzed simultaneously. Our RT-LAMP assay has a detection limit of 50 viral copies/reaction with the cycle threshold (Ct) value ≤ 39.7 ± 0.4 determined by the WHO-approved RT-qPCR assay. RT-LAMP-DETR exhibited a complete concordance with the results from naked-eye observation and RT-qPCR, achieving 100% sensitivity, specificity, and accuracy that altogether render it suitable for ultrasensitive point-of-care COVID-19 screening efforts. From the perspective of pandemic preparedness, our method offers a simpler, faster, and cheaper (∼$8/test) approach for COVID-19 testing and other emerging pathogens with respect to RT-qPCR.

Aquaculture Reports, 2015

Our previous work on the mechanism of virulence for the unique isolates of Vibrio parahaemolyticu... more Our previous work on the mechanism of virulence for the unique isolates of Vibrio parahaemolyticus that cause acute hepatopancreatic necrosis disease (VP AHPND) revealed that it was mediated by a binary Pir-like toxin pair ToxA and ToxB. These toxins are located on the pVA plasmid, a plasmid carried by AHPNDcausing strain of V. parahaemolyticus with a size of approximately 69 kbp. Using the coding sequences of ToxA, a one-step PCR detection method for VP AHPND was introduced in June 2014 but had the limitation that attempts to adapt it into a nested PCR protocol were unsuccessful. As a result, low levels of VP AHPND in shrimp or other samples could not be detected without first preparing an enrichment broth culture to allow bacterial growth before extraction of template DNA. Here, we describe the AP4 (abbreviation of AHPND detection version 4) method, a two-tube nested PCR method that targets the tandem genes ToxA and ToxB, including the 12 bp spacer that separates them on pVA plasmid. Testing of the method revealed that it gave 100% positive and negative predictive values for VP AHPND using a panel of 104 bacterial isolates including 51 VP AHPND isolates and 53 non-AHPND isolates, the latter including 34 isolates of V. parahaemolyticus and 19 isolates of other bacteria found in shrimp ponds, including other Vibrio species. The AP4 nested PCR method was 100 times more sensitive (100 fg total DNA template) than the one-step AP3 (10 pg total DNA template) method, and it could detect VP AHPND in experimentally challenged shrimp by 6 h post immersion (n = 2/3), while AP3 could not detect is until 12 h post immersion (n = 1/3). Thus, the AP4 method may be useful in detecting VP AHPND isolates in samples where target material is limited (e.g., small tissue quantity or archived DNA) and enrichment cannot be employed (i.e., frozen samples or samples preserved in alcohol).

Journal of fish diseases, 2021

Tilapia is one of the major aquaculture species with a global economic significance. Despite a hi... more Tilapia is one of the major aquaculture species with a global economic significance. Despite a high scale of production worldwide, mortality in many tilapia cultures has recently become a problem concerned with not only intensive farming but also the prevalence of infectious pathogens. Tilapia lake virus (TiLV) has emerged as a serious single-stranded RNA disease agent that thus far has continued to cause a number of incidences across the continents. Conventional PCR-based molecular detection techniques, despite having high sensitivity for TiLV, are not best suited for the onsite identification of infected fish mainly due to their requirement of laboratory resources and extended assay turnaround time. To address this practical limitation, we have developed a novel colorimetric assay based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and gold nanoparticle (AuNP)-labelled oligonucleotide reporter probe targeting the viral genomic segment 9 that enables the...

Clostridium perfringens is a key anaerobic pathogen causing food poisoning. Definitive detection ... more Clostridium perfringens is a key anaerobic pathogen causing food poisoning. Definitive detection by standard culture method is time-consuming and labor intensive. Current rapid commercial test kits are prohibitively expensive. It is thus necessary to develop rapid and cost-effective detection tool. Here, loop-mediated isothermal amplification (LAMP) in combination with a lateral-flow biosensor (LFB) was developed for visual inspection of C. perfringens-specific cpa gene. The specificity of the developed test was evaluated against 40 C. perfringens and 35 other bacterial strains, which showed no cross-reactivity, indicating 100% inclusivity and exclusivity. LAMP-LFB detection limit for artificially contaminated samples after enrichment for 16 h was 1–10 CFU/g sample, which was comparable to the commercial real-time PCR kit. The detection performance of LAMP-LFB was also compared to culture-based method using 95 food samples, which revealed the sensitivity (SE), specificity (SP) and C...

Diseases of Aquatic Organisms, May 1, 2009

Virus Research, 2008

Yellow head virus (YHV) is a pathogen of the black tiger shrimp (Penaeus monodon) and, with gilla... more Yellow head virus (YHV) is a pathogen of the black tiger shrimp (Penaeus monodon) and, with gillassociated virus (GAV), is one of two known invertebrate nidoviruses. We describe sequences of the large replicase gene (ORF1a) and 5 -and 3 -terminal UTRs, completing the 26,662 nt sequence of the YHV genome. ORF1a (12,219 nt) encodes a ∼462,662 Da polypeptide containing a putative 3C-like protease and a putative papain-like protease with the canonical C/H catalytic dyad and ␣ + ␤ fold. The read-through pp1ab polyprotein contains putative uridylate-specific endoribonuclease and ribose-2 -O-methyl transferase domains, and an exonuclease domain incorporating unusual dual Zn 2+ -binding fingers. Upstream of ORF1a, the 71 nt 5 -UTR shares 82.4% identity with the 68 nt 5 -UTR of GAV. The 677 nt 3 -terminal region contains a single 60 nt ORF, commencing 298 nt downstream of ORF3, that is identical to N-terminal coding region of the 249 nt GAV ORF4. Northern blots using RNA from YHV-infected shrimp and probes directed at ORF1a, ORF1b, ORF2 and ORF3 identified a nested set of 3 -coterminal RNAs comprising the full-length genomic RNA and two sub-genomic (sg) mRNAs. Intergenic sequences upstream of ORF2 and ORF3 share high identity with GAV, particularly in the conserved domains predicted to mediate sgmRNA transcription.

Virus Genes, 2009

Chimeric reporter genes were generated comprising nine different promoters of the white spot synd... more Chimeric reporter genes were generated comprising nine different promoters of the white spot syndrome virus linked to luciferase, with the aim to compare their transcriptional activities in insect cells. The promoters included the four non-structural genes DNA polymerase, ribonucleotide reductase small subunit, ribonucleotide reductase large subunit, and thymidine-thymidylate kinase, and the five structural genes VP15, VP19, VP24, VP26, and VP28. The promoters of the non-structural but not the structural genes can function in these cells, indicating that transcription of the non-structural genes can be recognized by cellular transcriptional machineries. While the structural genes were highly expressed in natural host cells during white spot syndrome virus infection, their promoters failed to direct transcription in insect cells, suggesting that transcription of these late genes may require virally induced host factor(s). Motifs essential for transcription of the above non-structural genes were identified by transient transfection of insect cells with constructs containing a series of deletions in the 50 terminal region and within the promoter. The minimal promoter sequences of these four genes were also capable of driving expression of the enhanced green fluorescent protein in insect cells.

Diseases of Aquatic Organisms, 2009

Aquaculture Reports, 2015

Talanta, 2022

Colorimetric loop-mediated DNA isothermal amplification-based assays have gained momentum in the ... more Colorimetric loop-mediated DNA isothermal amplification-based assays have gained momentum in the diagnosis of COVID-19 owing to their unmatched feasibility in low-resource settings. However, the vast majority of them are restricted to proprietary pH-sensitive dyes that limit downstream assay optimization or hinder efficient result interpretation. To address this problem, we developed a novel dual colorimetric RT-LAMP assay using in-house pH-dependent indicators to maximize the visual detection and assay simplicity, and further integrated it with the artificial intelligence (AI) operated tool (RT-LAMP-DETR) to enable a more precise and rapid result analysis in large scale testing. The dual assay leverages xylenol orange (XO) and a newly formulated lavender green (LG) dye for distinctive colorimetric readouts, which enhance the test accuracy when performed and analyzed simultaneously. Our RT-LAMP assay has a detection limit of 50 viral copies/reaction with the cycle threshold (Ct) value ≤ 39.7 ± 0.4 determined by the WHO-approved RT-qPCR assay. RT-LAMP-DETR exhibited a complete concordance with the results from naked-eye observation and RT-qPCR, achieving 100% sensitivity, specificity, and accuracy that altogether render it suitable for ultrasensitive point-of-care COVID-19 screening efforts. From the perspective of pandemic preparedness, our method offers a simpler, faster, and cheaper (∼$8/test) approach for COVID-19 testing and other emerging pathogens with respect to RT-qPCR.

Aquaculture Reports, 2015

Our previous work on the mechanism of virulence for the unique isolates of Vibrio parahaemolyticu... more Our previous work on the mechanism of virulence for the unique isolates of Vibrio parahaemolyticus that cause acute hepatopancreatic necrosis disease (VP AHPND) revealed that it was mediated by a binary Pir-like toxin pair ToxA and ToxB. These toxins are located on the pVA plasmid, a plasmid carried by AHPNDcausing strain of V. parahaemolyticus with a size of approximately 69 kbp. Using the coding sequences of ToxA, a one-step PCR detection method for VP AHPND was introduced in June 2014 but had the limitation that attempts to adapt it into a nested PCR protocol were unsuccessful. As a result, low levels of VP AHPND in shrimp or other samples could not be detected without first preparing an enrichment broth culture to allow bacterial growth before extraction of template DNA. Here, we describe the AP4 (abbreviation of AHPND detection version 4) method, a two-tube nested PCR method that targets the tandem genes ToxA and ToxB, including the 12 bp spacer that separates them on pVA plasmid. Testing of the method revealed that it gave 100% positive and negative predictive values for VP AHPND using a panel of 104 bacterial isolates including 51 VP AHPND isolates and 53 non-AHPND isolates, the latter including 34 isolates of V. parahaemolyticus and 19 isolates of other bacteria found in shrimp ponds, including other Vibrio species. The AP4 nested PCR method was 100 times more sensitive (100 fg total DNA template) than the one-step AP3 (10 pg total DNA template) method, and it could detect VP AHPND in experimentally challenged shrimp by 6 h post immersion (n = 2/3), while AP3 could not detect is until 12 h post immersion (n = 1/3). Thus, the AP4 method may be useful in detecting VP AHPND isolates in samples where target material is limited (e.g., small tissue quantity or archived DNA) and enrichment cannot be employed (i.e., frozen samples or samples preserved in alcohol).

Journal of fish diseases, 2021

Tilapia is one of the major aquaculture species with a global economic significance. Despite a hi... more Tilapia is one of the major aquaculture species with a global economic significance. Despite a high scale of production worldwide, mortality in many tilapia cultures has recently become a problem concerned with not only intensive farming but also the prevalence of infectious pathogens. Tilapia lake virus (TiLV) has emerged as a serious single-stranded RNA disease agent that thus far has continued to cause a number of incidences across the continents. Conventional PCR-based molecular detection techniques, despite having high sensitivity for TiLV, are not best suited for the onsite identification of infected fish mainly due to their requirement of laboratory resources and extended assay turnaround time. To address this practical limitation, we have developed a novel colorimetric assay based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and gold nanoparticle (AuNP)-labelled oligonucleotide reporter probe targeting the viral genomic segment 9 that enables the...

Clostridium perfringens is a key anaerobic pathogen causing food poisoning. Definitive detection ... more Clostridium perfringens is a key anaerobic pathogen causing food poisoning. Definitive detection by standard culture method is time-consuming and labor intensive. Current rapid commercial test kits are prohibitively expensive. It is thus necessary to develop rapid and cost-effective detection tool. Here, loop-mediated isothermal amplification (LAMP) in combination with a lateral-flow biosensor (LFB) was developed for visual inspection of C. perfringens-specific cpa gene. The specificity of the developed test was evaluated against 40 C. perfringens and 35 other bacterial strains, which showed no cross-reactivity, indicating 100% inclusivity and exclusivity. LAMP-LFB detection limit for artificially contaminated samples after enrichment for 16 h was 1–10 CFU/g sample, which was comparable to the commercial real-time PCR kit. The detection performance of LAMP-LFB was also compared to culture-based method using 95 food samples, which revealed the sensitivity (SE), specificity (SP) and C...

Diseases of Aquatic Organisms, May 1, 2009

Virus Research, 2008

Yellow head virus (YHV) is a pathogen of the black tiger shrimp (Penaeus monodon) and, with gilla... more Yellow head virus (YHV) is a pathogen of the black tiger shrimp (Penaeus monodon) and, with gillassociated virus (GAV), is one of two known invertebrate nidoviruses. We describe sequences of the large replicase gene (ORF1a) and 5 -and 3 -terminal UTRs, completing the 26,662 nt sequence of the YHV genome. ORF1a (12,219 nt) encodes a ∼462,662 Da polypeptide containing a putative 3C-like protease and a putative papain-like protease with the canonical C/H catalytic dyad and ␣ + ␤ fold. The read-through pp1ab polyprotein contains putative uridylate-specific endoribonuclease and ribose-2 -O-methyl transferase domains, and an exonuclease domain incorporating unusual dual Zn 2+ -binding fingers. Upstream of ORF1a, the 71 nt 5 -UTR shares 82.4% identity with the 68 nt 5 -UTR of GAV. The 677 nt 3 -terminal region contains a single 60 nt ORF, commencing 298 nt downstream of ORF3, that is identical to N-terminal coding region of the 249 nt GAV ORF4. Northern blots using RNA from YHV-infected shrimp and probes directed at ORF1a, ORF1b, ORF2 and ORF3 identified a nested set of 3 -coterminal RNAs comprising the full-length genomic RNA and two sub-genomic (sg) mRNAs. Intergenic sequences upstream of ORF2 and ORF3 share high identity with GAV, particularly in the conserved domains predicted to mediate sgmRNA transcription.

Virus Genes, 2009

Chimeric reporter genes were generated comprising nine different promoters of the white spot synd... more Chimeric reporter genes were generated comprising nine different promoters of the white spot syndrome virus linked to luciferase, with the aim to compare their transcriptional activities in insect cells. The promoters included the four non-structural genes DNA polymerase, ribonucleotide reductase small subunit, ribonucleotide reductase large subunit, and thymidine-thymidylate kinase, and the five structural genes VP15, VP19, VP24, VP26, and VP28. The promoters of the non-structural but not the structural genes can function in these cells, indicating that transcription of the non-structural genes can be recognized by cellular transcriptional machineries. While the structural genes were highly expressed in natural host cells during white spot syndrome virus infection, their promoters failed to direct transcription in insect cells, suggesting that transcription of these late genes may require virally induced host factor(s). Motifs essential for transcription of the above non-structural genes were identified by transient transfection of insect cells with constructs containing a series of deletions in the 50 terminal region and within the promoter. The minimal promoter sequences of these four genes were also capable of driving expression of the enhanced green fluorescent protein in insect cells.

Diseases of Aquatic Organisms, 2009

Aquaculture Reports, 2015