Detection of Salmonella DNA and drug-resistance mutation by PCR-based CRISPR-lbCas12a system

Abstract

Abstract Salmonella is an important foodborne pathogen, which can cause serious public health problems. Rapid and accurate detection of Salmonella infection and drug resistance mutations in patients will provide timely guidance for clinical treatment and avoid disease progression and other related clinical problems. Here, we established a highly sensitive and quick method for Salmonella and drug resistance mutation detection based on polymerase chain reaction (PCR) and CRISPR-lbCas12a system and evaluated its practicability with clinical samples. Specific CRISPR RNAs (crRNAs) and primers are designed for Salmonella DNA and parC gene S80I mutation diagnosis. CrRNAs with and without phosphorylated modification and different crRNA preparation methods are used to assess the effect on the detection system. After optimization, we detected as low as one copy of Salmonella DNA and drug resistance mutation parC S80I with the Salmonella DNA standard. For 94 clinical samples, this method also showed high sensitivity (100%, 95% CI: 84.98–100%) and specificity (98.48%, 95% CI: 90.73–99.92%) with less time (3 h) than plate culture (16 h) and conventional antimicrobial susceptibility testing (over 16 h). Besides, one parC S80I mutant strain was detected, which is consistent with the result of DNA sequencing. Taken together, we established a highly sensitive and specific method for Salmonella infection and parC S80I drug resistance mutation detection with fewer reagents and ordinary instruments. This assay has wide application prospects for fast detection of pathogen (bacterium and virus) infection, drug resistance determination, and proper treatment guidance.

Keywords

• Salmonella
• Drug resistance mutation
• CRISPR-lbCas12a
• PCR