Rapid, ultrasensitive and highly specific diagnosis of Mycoplasma pneumoniae by a CRISPR-based detection platform

Abstract

Mycoplasma pneumoniae (MP) is an important causative agent of morbidity and mortality among all age groups, especially among patients of extreme ages. Improved and readily available tests for accurate, sensitive and rapid diagnosis of MP infection is sorely needed. Here, we developed a CRISPR-Cas12b-based detection platform on the basis of recombinase polymerase amplification (RPA) for rapid, simple, and accurate diagnosis of MP infection, named MP-RPA-CRISPR. The RPA was employed for amplifying the community-acquired respiratory distress syndrome (CARDS) toxin gene of MP strains at the optimal reaction temperature 37°C. The resulting amplicons were decoded by the CRISPR-Cas12b-based detection platform, which was interpreted by real-time PCR system and by naked eye under blue light. The MP-RPA-CRISPR can detected down to 5 fg of genomic DNA templates of MP strains and accurately distinguish MP strains from non-MP strains without any cross-reactivity. A total of 96 bronchoalveolar lavage fluid (BALF)samples collected from patients suspected of respiratory infection were used to evaluate the clinical performance of the MP-RPA-CRISPR assay. As a result, our assay accurately diagnosed 45 MP-infected samples and 51 non-MP infected sample, and the results obtained from MP-RPA-CRISPR were consistent with microfluidic chip technology. In conclusion, our MP-RPA-CRISPR assay is a simple, rapid, portable and highly sensitive method to diagnose MP infection, which can be used as a promising tool in a variety of settings including clinical, field, and resource-limited aeras.

Keywords: CRISPR; Cas12b; MP infection; Mycoplasma pneumoniae; recombinase polymerase amplification.

Figure 1

Schematic illustration of MP-RPA-CRISPR assay’s principle. (A) Principle of RPA assay. After RPA reaction, a plenty of amplicons containing PAM site specific for CRISPR-Cas12b (TTG) were produced. (B) Principle and procedure of the CRISPR-Cas12b-based trans-cleavage detection. After the pre-amplification by RPA, the PAM site within the resultant amplicons was recognized by the CRISPR-Cas12b/gRNA complex and formed a ternary complex, which was then activated and nonspecifically cut the FAM/BHQ1 labeled probe, resulting to the release of fluorescent signals. (C) Workflow of the MP-RPA-CRISPR assay. The whole assay included four steps, including DNA extraction, RPA reaction, CRISPR-Casa12b tans-cleavage and results reporting. The interpretation of results reporting could be performed by both real-time PCR system and by naked eye under blue light.

Figure 2

Confirmation and validation of the MP-RPA-CRISPR assay. (A) Confirmation of the amplification products of RPA by agarose gel electrophoresis (AGE) method. (B) Confirmation of the amplification products of RPA using CRISPR-Cas12b-based detection platform by naked eye under blue light. (C) Confirmation of the amplification products of RPA using CRISPR-Cas12b-based detection platform by real-time PCR system. Templates of 1-3 were the genomic DNA of MP, KP and DW, respectively. M represented the 100 bp DNA marker.

Figure 3

Sensitivity of MP-RPA-CRISPR assay. (A) Sensitivity of the MP-RPA assay detected by AGE method. (B) Sensitivity of the MP-RPA-CRISPR assay visualized by naked eye under blue light. (C), Sensitivity of the MP-RPA-CRISPR assay recorded by the real-time PCR system. 1–9 represented the genomic DNA templates of MP strain M129 ranging from 5 ng to 0.5 fg and that of Kp. The LoD level of MP-RPA-CRISPR assay was 5 fg per reaction, and that for RPA assay visualized by AGE was 50 fg per reaction.

Figure 4

Specificity of the MP-RPA-CRISPR assay. (A) Specificity of the MP-RPA assay detected by AGE method. (B) Specificity of the MP-RPA-CRISPR assay visualized by naked eye under blue light. (C) Specificity of the MP-RPA-CRISPR assay recorded by the real-time PCR system. 1 represented the genomic DNA templates of MP strain M129, 2-27 represented the genomic DNA templates of 25 non-MP pathogens and DW. Only the genomic DNA of the MP strain M129 was positively detected by the MP-RPA-CRISPR assay.

Figure 5

Clinical feasibility validation of the MP-RPA-CRISPR assay. A total of 96 clinical samples were used to confirm the clinical performance of the MP-RPA-CRISPR assay. All the positive samples that tested by microfluidic chip technology showed positive results when detected by the MP-RPA-CRISPR assay, and the negative ones displayed negative results as well. S1-S96 represented the 96 bronchoalveolar lavage fluid (BALF) samples collected from patients suspected of respiratory infection based on clinical symptoms.