Detection of Burkholderia pseudomallei with CRISPR-Cas12a based on specific sequence tags

Abstract

Melioidosis is a bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei), posing a significant threat to public health. Rapid and accurate detection of B. pseudomallei is crucial for preventing and controlling melioidosis. However, identifying B. pseudomallei is challenging due to its high similarity to other species in the same genus. To address this issue, this study proposed a dual-target method that can specifically identify B. pseudomallei in less than 40 min. We analyzed 1722 B. pseudomallei genomes to construct large-scale pan-genomes and selected specific sequence tags in their core genomes that effectively distinguish B. pseudomallei from its closely related species. Specifically, we selected two specific tags, LC1 and LC2, which we combined with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas12a) system and recombinase polymerase amplification (RPA) pre-amplification. Our analysis showed that the dual-target RPA-CRISPR/Cas12a assay has a sensitivity of approximately 0.2 copies/reaction and 10 fg genomic DNA for LC1, and 2 copies/reaction and 20 fg genomic DNA for LC2. Additionally, our method can accurately and rapidly detect B. pseudomallei in human blood and moist soil samples using the specific sequence tags mentioned above. In conclusion, the dual-target RPA-CRISPR/Cas12a method is a valuable tool for the rapid and accurate identification of B. pseudomallei in clinical and environmental samples, aiding in the prevention and control of melioidosis.

Rapid detection of Pseudomonas aeruginosa by recombinase polymerase amplification combined with CRISPR-Cas12a biosensing system

“…The CRISPR-Cas biosensing system could transfer the sequence information of targets to detectable signals (such as fluorescence or colorimetric values) by employing the collateral cleavage activities of the Cas effectors (Cas12a, Cas12b, Cas13, and Cas14), conferring this technology high sensitivity and specificity of detection and simplicity to develop, which also exhibits great potential in point-of-care tests ( Gootenberg et al., 2018 ; Myhrvold et al., 2018 ; Bonini et al., 2021 ; Jirawannaporn et al., 2022 ; Kumaran et al., 2023 ; Zheng et al., 2023 ). In particular, by coupling isothermal amplification procedure, the detection performance of CRISPR-Cas biosensing system is greatly improved, and the target type also can be converted ( Li et al., 2019 ; Zhang et al., 2023 ). Recently, the CRISPR-Cas–based biosensing detection platforms, such as SHERLOCK (Specific High Sensitivity Enzymatic Reporter Unlocking, RPA combination with Cas13a) ( Myhrvold et al., 2018 ) and DETECTR (DNA Endonuclear Targeted Crispr Trans Reporter, RPA combination with Cas12a) ( Chen et al., 2018 ), have been rapidly developed and already commercial available for pathogen detection.…”

Recombinase polymerase amplification: method’s characteristics and applications in diagnostics of infectious diseases

“…SHERLOCK. Одним из наиболее активно развивающихся методов амплификации НК является «Specifc High-Sensitivity Enzymatic Reporter На основе системы CRISPR-Cas разработаны различные диагностические платформы для многих инфекционных патогенов, предложены разнообразные системы пробоподготовки: от применения термического лизиса в качестве экстракции НК до обработки магнитными частицами с целью накопления искомого материала и повышения чувствительности теста [25][26][27]. По данным публикаций, метод SHERLOCK обладает более высокой чувствительностью по сравнению с аналогичными подходами, предел обнаружения сочетанного использования технологий РПА и CRISPR-Cas составляет несколько копий мишени в реакции [24,28].…”

“…В ряде исследований была показана высокая устойчивость РПА к присутствию ингибиторов. При проведении термического лизиса реакцию можно проводить без полноценного выделения НК из таких образцов, как сыворотка, кал, моча, плевральная жидкость, молоко, почва [17,27,[41][42][43][44]. Продемонстрирована устойчивость к наличию гемоглобина, этанола и гепарина [45].…”