CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus
<i>Orientia tsutsugamushi</i> is responsible for causing scrub typhus (ST) and is the leading cause of acute encephalitis syndrome (AES) in AES patients. A rapid and sensitive method to detect scrub typhus on-site is essential for the timely deployment of control measures. In the current...
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| Format: | Article |
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MDPI AG
2023-12-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/13/12/1021 |
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| author | Pooja Bhardwaj Nikita Shrikant Nanaware Sthita Pragnya Behera Smita Kulkarni Hirawati Deval Rajesh Kumar Gaurav Raj Dwivedi Rajni Kant Rajeev Singh |
| author_facet | Pooja Bhardwaj Nikita Shrikant Nanaware Sthita Pragnya Behera Smita Kulkarni Hirawati Deval Rajesh Kumar Gaurav Raj Dwivedi Rajni Kant Rajeev Singh |
| author_sort | Pooja Bhardwaj |
| collection | DOAJ |
| description | <i>Orientia tsutsugamushi</i> is responsible for causing scrub typhus (ST) and is the leading cause of acute encephalitis syndrome (AES) in AES patients. A rapid and sensitive method to detect scrub typhus on-site is essential for the timely deployment of control measures. In the current study, we developed a rapid, sensitive, and instrument-free lateral flow assay (LFA) detection method based on CRISPR/Cas12a technology for diagnosing ST (named LoCIST). The method is completed in three steps: first, harnessing the ability of recombinase polymerase for isothermal amplification of the target gene; second, CRISPR/Cas12a-based recognition of the target; and third, end-point detection by LFA. The detection limit of LoCIST was found to be one gene copy of ST genomic DNA per reaction, and the process was complete within an hour. In 81 clinical samples, the assay showed no cross-reactivity with other rickettsial DNA and was 100% consistent with PCR detection of ST. LoCIST demonstrated 97.6% sensitivity and 100% specificity. Overall, the LoCIST offers a novel alternative for the portable, simple, sensitive, and specific detection of ST, and it may help prevent and control AES outbreaks due to ST. In conclusion, LoCIST does not require specialized equipment and poses a potential for future applications as a point-of-care diagnostic. |
| first_indexed | 2024-03-08T20:57:01Z |
| format | Article |
| id | doaj.art-b1f2cce6d3a3484596e65e8525785352 |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-08T20:57:01Z |
| publishDate | 2023-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-b1f2cce6d3a3484596e65e85257853522023-12-22T13:56:23ZengMDPI AGBiosensors2079-63742023-12-011312102110.3390/bios13121021CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub TyphusPooja Bhardwaj0Nikita Shrikant Nanaware1Sthita Pragnya Behera2Smita Kulkarni3Hirawati Deval4Rajesh Kumar5Gaurav Raj Dwivedi6Rajni Kant7Rajeev Singh8ICMR-Regional Medical Research Centre Gorakhpur, BRD Medical College Campus, Gorakhpur 273013, IndiaICMR-National AIDS Research Institute, Bhosari, Pune 411026, IndiaICMR-Regional Medical Research Centre Gorakhpur, BRD Medical College Campus, Gorakhpur 273013, IndiaICMR-National AIDS Research Institute, Bhosari, Pune 411026, IndiaICMR-Regional Medical Research Centre Gorakhpur, BRD Medical College Campus, Gorakhpur 273013, IndiaRGSC, Department of Genetics and Plant Breeding, Banaras Hindu University, Varanasi 221005, IndiaICMR-Regional Medical Research Centre Gorakhpur, BRD Medical College Campus, Gorakhpur 273013, IndiaICMR-Regional Medical Research Centre Gorakhpur, BRD Medical College Campus, Gorakhpur 273013, IndiaICMR-Regional Medical Research Centre Gorakhpur, BRD Medical College Campus, Gorakhpur 273013, India<i>Orientia tsutsugamushi</i> is responsible for causing scrub typhus (ST) and is the leading cause of acute encephalitis syndrome (AES) in AES patients. A rapid and sensitive method to detect scrub typhus on-site is essential for the timely deployment of control measures. In the current study, we developed a rapid, sensitive, and instrument-free lateral flow assay (LFA) detection method based on CRISPR/Cas12a technology for diagnosing ST (named LoCIST). The method is completed in three steps: first, harnessing the ability of recombinase polymerase for isothermal amplification of the target gene; second, CRISPR/Cas12a-based recognition of the target; and third, end-point detection by LFA. The detection limit of LoCIST was found to be one gene copy of ST genomic DNA per reaction, and the process was complete within an hour. In 81 clinical samples, the assay showed no cross-reactivity with other rickettsial DNA and was 100% consistent with PCR detection of ST. LoCIST demonstrated 97.6% sensitivity and 100% specificity. Overall, the LoCIST offers a novel alternative for the portable, simple, sensitive, and specific detection of ST, and it may help prevent and control AES outbreaks due to ST. In conclusion, LoCIST does not require specialized equipment and poses a potential for future applications as a point-of-care diagnostic.https://www.mdpi.com/2079-6374/13/12/1021scrub typhus<i>Orientia tsutsugamushi</i>rapid diagnostic kitGorakhpuracute encephalitis syndrome56 kDa gene |
| spellingShingle | Pooja Bhardwaj Nikita Shrikant Nanaware Sthita Pragnya Behera Smita Kulkarni Hirawati Deval Rajesh Kumar Gaurav Raj Dwivedi Rajni Kant Rajeev Singh CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus Biosensors scrub typhus <i>Orientia tsutsugamushi</i> rapid diagnostic kit Gorakhpur acute encephalitis syndrome 56 kDa gene |
| title | CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus |
| title_full | CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus |
| title_fullStr | CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus |
| title_full_unstemmed | CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus |
| title_short | CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus |
| title_sort | crispr cas12a based detection platform for early and rapid diagnosis of scrub typhus |
| topic | scrub typhus <i>Orientia tsutsugamushi</i> rapid diagnostic kit Gorakhpur acute encephalitis syndrome 56 kDa gene |
| url | https://www.mdpi.com/2079-6374/13/12/1021 |
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