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

<strong>Background: </strong><em>Streptococcus agalactiae</em> or group B <em>Streptococcus</em> (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). <strong>Methods: </strong>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. <strong>Results:</strong> The CRISPR-GBS assay can be completed within 35 minutes 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. <strong>Conclusions:</strong> 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.

背景：无乳链球菌（Streptococcus agalactiae），又称B群链球菌（GBS），是引发新生儿发病与死亡的主要感染性病原体之一。对于存在胎膜早破（premature rupture of membrane, PROM）的孕妇，建立快速、超灵敏且稳健的GBS检测方法至关重要。方法：本研究开发了一种CRISPR-GBS检测方法，该方法结合了重组酶聚合酶扩增（recombinase polymerase amplification, RPA）与CRISPR/Cas12a系统的优势，用于GBS检测。研究采用179例胎膜早破孕妇的阴道或宫颈拭子样本，对CRISPR-GBS检测方法的临床性能进行评估，并与基于培养的基质辅助激光解吸电离飞行时间质谱（culture-MS）法及实时定量聚合酶链反应（qPCR）法进行平行对照。结果：CRISPR-GBS检测可在35分钟内完成，检测下限低至5拷贝·μL⁻¹。与culture-MS法相比，CRISPR-GBS检测的灵敏度为96.64%（144/149，95%置信区间[CI] = 92.39–98.56%），特异性为100%（30/30，95%置信区间[CI] = 88.65–100%）；该方法与qPCR法的符合率高达98.88%。结论：本研究建立的CRISPR-GBS检测平台可实现快速、准确、易操作且成本效益优异的GBS检测，为产时GBS定植筛查提供了极具应用前景的工具。