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.

Keywords: Burkholderia pseudomallei; CRISPR-Cas12a; species discrimination; specific sequence tags; visual detection.

Figure 1

Workflow of this project. (A) Acquisition of B. pseudomallei-specific tags. (B) Schema of assay process for the detection of B. pseudomallei with the dual–target RPA–CRISPR/Cas12a assay.

Figure 2

Pan-genome analysis of B. pseudomallei and B. mallei. (A) Number of gene families (B. pseudomallei and B. mallei). (B) The trend chart of the size of core-pan genes (B. pseudomallei and B. mallei).

Figure 3

The genome annotations of B. pseudomallei K96243. This includes, from outer to inner rings, the distribution of the 2 B. pseudomallei-specific tags (for subsequent CRISPR-Cas12a analysis), the contigs, CDS on the forward strand, CDS on the reverse strand, RNA genes, CDS with homology to known antimicrobial resistance genes, CDS with homology to known virulence factors, GC content, and GC skew.

Figure 4

Screening of optimal RPA primers and crRNAs. (A) LC1 RPA primer screening using the same concentration of DNA template. (B) LC2 RPA primer screening using the same concentration of DNA template. (C) Identifying the best LC1 RPA primer and crRNA. (D) Identifying the best LC2 RPA primer and crRNA.

Figure 5

Sensitivity evaluation of the dual-target RPA-CRISPR/Cas12a assay. (A,B) Positive reference plasmids and (C,D) B. pseudomallei genomic DNA, LC1 RPA-CRISPR/Cas12a assay. (E,F) Positive reference plasmids and (G,H) B. pseudomallei genomic DNA, LC2 RPA-CRISPR/Cas12a assay. Error bars represent mean ± SEM, where n = 5 replicates, matched samples t-test, ****(p < 0.0001); (A) **(1 copies/reaction, p = 0.0015; 0.2 copies/reaction, p = 0.0050); (C) **(10 fg, p = 0.0054); ns (non-significant).

Figure 6

Specificity evaluation of dual-target RPA-CRISPR/Cas12a assay, Error bars represent mean ± SEM, where n = 4 replicates, matched samples t-test, ****(p < 0.0001), ns (non-significant), LC1 (A,C) and LC2 (B,D).

Figure 7

Blood and moist soil sample tests by the dual-target RPA-CRISPR/Cas12a assay and the RT-PCR assay, concentration of DNA, C_DNA. (A) Blood samples test. (B) Moist soil samples test. (C) Analyze the potential cross-reacting bacterial DNA in the simulation experiment. (D) The RT-PCR assay.