Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a
Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those...
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ftdoajarticles:oai:doaj.org/article:7338081e78dc4217933d90e602999800 2023-05-15T15:16:43+02:00 Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a Somsakul Pop Wongpalee Hathairat Thananchai Claire Chewapreecha Henrik B. Roslund Chalita Chomkatekaew Warunya Tananupak Phumrapee Boonklang Sukritpong Pakdeerat Rathanin Seng Narisara Chantratita Piyawan Takarn Phadungkiat Khamnoi 2022-08-01T00:00:00Z https://doaj.org/article/7338081e78dc4217933d90e602999800 EN eng Public Library of Science (PLoS) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9423629/?tool=EBI https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 https://doaj.org/article/7338081e78dc4217933d90e602999800 PLoS Neglected Tropical Diseases, Vol 16, Iss 8 (2022) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2022 ftdoajarticles 2022-12-30T20:34:52Z Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify B. pseudomallei DNA from clinical isolates. Through in silico search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to B. pseudomallei, named crBP34. The crBP34-based detection assay can detect as few as 40 copies of B. pseudomallei genomic DNA while discriminating against other tested common pathogens. When coupled with a lateral flow dipstick, the assay readout can be simply performed without the loss of sensitivity and does not require expensive equipment. This crBP34-based detection assay provides high sensitivity, specificity and simple detection method for B. pseudomallei DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA. Author summary Melioidosis is a fatal infectious disease caused by a Gram-negative bacterium called Burkholderia pseudomallei. The bacteria can be found in many parts of the world, especially in the tropical and subtropical regions. Infection displays a variety of symptoms such as pneumonia, organ abscess and septicemia. The latter can lead to death within 24–48 hours if not properly diagnosed and treated. Rapid and accurate diagnosis, consequently, are essential for saving patients’ lives. Currently, culturing B. pseudomallei is a gold standard diagnostic method, but the assay turnaround time is 2–4 days, and the result could be of low sensitivity. Other detection methods such as real-time PCR and serological assays are limited by availability of equipment and by low specificity in endemic areas, respectively. For these reasons, in this study we developed a ... Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic |
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Directory of Open Access Journals: DOAJ Articles |
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English |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Somsakul Pop Wongpalee Hathairat Thananchai Claire Chewapreecha Henrik B. Roslund Chalita Chomkatekaew Warunya Tananupak Phumrapee Boonklang Sukritpong Pakdeerat Rathanin Seng Narisara Chantratita Piyawan Takarn Phadungkiat Khamnoi Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify B. pseudomallei DNA from clinical isolates. Through in silico search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to B. pseudomallei, named crBP34. The crBP34-based detection assay can detect as few as 40 copies of B. pseudomallei genomic DNA while discriminating against other tested common pathogens. When coupled with a lateral flow dipstick, the assay readout can be simply performed without the loss of sensitivity and does not require expensive equipment. This crBP34-based detection assay provides high sensitivity, specificity and simple detection method for B. pseudomallei DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA. Author summary Melioidosis is a fatal infectious disease caused by a Gram-negative bacterium called Burkholderia pseudomallei. The bacteria can be found in many parts of the world, especially in the tropical and subtropical regions. Infection displays a variety of symptoms such as pneumonia, organ abscess and septicemia. The latter can lead to death within 24–48 hours if not properly diagnosed and treated. Rapid and accurate diagnosis, consequently, are essential for saving patients’ lives. Currently, culturing B. pseudomallei is a gold standard diagnostic method, but the assay turnaround time is 2–4 days, and the result could be of low sensitivity. Other detection methods such as real-time PCR and serological assays are limited by availability of equipment and by low specificity in endemic areas, respectively. For these reasons, in this study we developed a ... |
format |
Article in Journal/Newspaper |
author |
Somsakul Pop Wongpalee Hathairat Thananchai Claire Chewapreecha Henrik B. Roslund Chalita Chomkatekaew Warunya Tananupak Phumrapee Boonklang Sukritpong Pakdeerat Rathanin Seng Narisara Chantratita Piyawan Takarn Phadungkiat Khamnoi |
author_facet |
Somsakul Pop Wongpalee Hathairat Thananchai Claire Chewapreecha Henrik B. Roslund Chalita Chomkatekaew Warunya Tananupak Phumrapee Boonklang Sukritpong Pakdeerat Rathanin Seng Narisara Chantratita Piyawan Takarn Phadungkiat Khamnoi |
author_sort |
Somsakul Pop Wongpalee |
title |
Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a |
title_short |
Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a |
title_full |
Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a |
title_fullStr |
Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a |
title_full_unstemmed |
Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a |
title_sort |
highly specific and sensitive detection of burkholderia pseudomallei genomic dna by crispr-cas12a |
publisher |
Public Library of Science (PLoS) |
publishDate |
2022 |
url |
https://doaj.org/article/7338081e78dc4217933d90e602999800 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 16, Iss 8 (2022) |
op_relation |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9423629/?tool=EBI https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 https://doaj.org/article/7338081e78dc4217933d90e602999800 |
_version_ |
1766347014102056960 |