CRISPR/Cas12a-based assay for the rapid and high-sensitivity detection of Streptococcus agalactiae colonization in pregnant women with premature rupture of membrane

Abstract

Background Streptococcus agalactiae or group B Streptococcus (GBS) is a leading infectious cause of neonatal morbidity and mortality. It is essential to establish a robust method for the rapid and ultra-sensitive detection of GBS in pregnant women with premature rupture of membrane (PROM). Methods This study developed a CRISPR-GBS assay that combined the advantages of the recombinase polymerase amplification (RPA) and CRISPR/Cas12a system for GBS detection. The clinical performance of the CRISPR-GBS assay was assessed using vaginal or cervical swabs that were collected from 179 pregnant women with PROM, compared in parallel to culture-based matrix-assisted laser desorption ionization time-of-flight mass spectrometry (culture-MS) method and real-time quantitative polymerase chain reaction (qPCR) assay. Results The CRISPR-GBS assay can be completed within 35 min and the limit of detection was as low as 5 copies μL−1. Compared with the culture-MS, the CRISPR-GBS assay demonstrated a sensitivity of 96.64% (144/149, 95% confidence interval [CI] 92.39–98.56%) and a specificity of 100% (30/30, 95% CI 88.65–100%). It also had a high concordance rate of 98.88% with the qPCR assay. Conclusions The established CRISPR-GBS platform can detect GBS in a rapid, accurate, easy-to-operate, and cost-efficient manner. It offered a promising tool for the intrapartum screening of GBS colonization.

Establishment and application of a rapid molecular diagnostic platform for the isothermal visual amplification of group B Streptococcus based on recombinase polymerase

“…et al, 2021). However, the widespread adoption of laboratory diagnosis for clinical GBS screening remains challenging, particularly in resource-limited areas (Yu et al, 2023). This study addresses this issue by employing RPA technology combined with nfo enzyme and LPS, enabling rapid, equipment-independent GBS detection suitable even in remote settings.…”

“…Detection results using different methods (Yu et al, 2023), our study focuses on improving specificity and visualization. We used an improved RPA amplification reagent with an nfo enzyme and a specific probe with a THF site.…”

“…This process requires no complex equipment and special experimental conditions, enabling equipment-independent GBS detection (Piepenburg et al, 2006). Although the basic RPA technique has been used for GBS detection, the interpretation of its results is still limited to wellequipped laboratories (Yu et al, 2023).…”

Combination of nucleic acid amplification and CRISPR/Cas technology in pathogen detection

“… Zhang et al (2022) established CRISPR/Cas12a combined with RPA to detect Aspergillus besseyi with a LOD of 1 copy/μL, which was combined with a lateral flow strip assay to visualize the results. Yu et al (2023) coarsely extracted DNA from vaginal or cervical swabs of pregnant women by heat lysis, then selected the highly conserved region of the cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor as a target for detection, and enriched the target sequences by amplification using RPA, and finally utilised the CRISPR/Cas12a system for target-specific identification and signal release( Figure 4A ). Our team Tian et al (2021) combined the RPA-Cas12a technique with lateral flow immunoassay, thus realizing the rapid detection of L. monocytogenes .…”

“… (A) Schematic diagram of the CRISPR-GBS assay ( Yu et al, 2023 ); (B) detection of HPV in patient samples on CRISPR ( Zhou et al, 2023 ). …”