# Application of CRISPR/Cas13a system on the rapid detection of Salmonella spp

## TL;DR

This study introduces a fast and accurate CRISPR-based test for detecting Salmonella, which could improve early diagnosis and outbreak control.

## Contribution

A novel CRISPR/Cas13a-based assay for rapid and sensitive Salmonella detection with potential for point-of-care use.

## Key findings

- The CRISPR/Cas13a-SE assay detected Salmonella in 60 minutes with a limit of detection of 100 fg/μL.
- The assay showed high concordance rates (98.79% to 100%) compared to traditional culture methods across multiple cohorts.
- The method is more sensitive than PCR and suitable for low bacterial load cases and asymptomatic carrier screening.

## Abstract

Salmonella spp. infections can manifest in various clinical symptoms, from asymptomatic carriage to gastroenteritis, and even severe sepsis. Given the rapid progression of the disease and its potential to cause severe outcomes or trigger cluster outbreaks, making the detection of Salmonella spp. critically important. Although broth enrichment culture is considered the gold standard, it is time-consuming and involves multiple steps, making it difficult to meet urgent diagnostic needs. Hence, prompt and precise detection of Salmonella spp. is crucial not only for early diagnosis and effective treatment, but also for preventing transmission, controlling outbreaks, and screening asymptomatic Salmonella carrier.

This study developed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) -SE assay that integrated the advantages of the recombinase polymerase amplification (RPA) and CRISPR/Cas13a system for detecting Salmonella spp. The clinical performance of CRISPR/Cas13a-SE assay was evaluated by a cohort of 94 inpatients with diarrhea and three prospective studies.

The CRISPR/Cas13a-SE assay can be completed within 60 minutes, and its limit of detection was 100 fg/μL. Compared to the broth enrichment culture, the CRISPR/Cas13a-SE assay demonstrated a sensitivity of 87.5% and a specificity of 98.8% in a cohort of 94 inpatients with diarrhea. In our prospective studies involved three distinct cohorts: 1,662 food handlers, 211 outpatients with diarrhea, and 154 inpatients with Gram-negative bacteremia. Compared with broth enrichment culture, CRISPR/Cas13a-SE assay had a high concordance rate of 98.79% (1,642/1,662), 99.52% (210/211), and 100.00% (154/154) respectively.

We demonstrated that the CRISPR/Cas13a-SE system showed excellent detection performance for infectious diarrhea caused by Salmonella spp. The combined use of CRISPR/Cas13a-SE with the blood culture method enhances the rapid diagnosis of invasive salmonellosis, which is crucial for early target-based therapy. Additionally, screening of asymptomatic Salmonella carrier will be benefit for disease prevention and control.

Prompt and precise detection of Salmonella spp. is crucial not only for early diagnosis and effective treatment, but also for preventing transmission, controlling outbreaks, and screening asymptomatic Salmonella carrier. In this study, we established a novel CRISPR-based method for Salmonella spp. rapid diagnosis. Our findings indicate that CRISPR/Cas13a-SE is more sensitive, particularly in cases of low bacterial load, and is simpler and faster than the PCR method. Its potential for cost-effective development, simplicity, and convenience suggests that CRISPR/Cas13a-SE could be developed into a low-cost point-of-care testing (POCT) device, utilizing basic fluorescence-detecting equipment to make the process faster and more accessible.