Liang Jia-Rui, Yan Shu-Ning, Yang Han-Yin, Yang Shuo, Shen Yu-Juan, Huo Le-Le, Cai Yu-Chun, Mo Zi-Ran, Zheng Bin, Xu Bin, Hu Wei
National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute of Parasitic Diseases at Chinese Center for Disease Control and Prevention, Chinese Center for Tropical Diseases Research, Shanghai, China.
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Chinese Center for Tropical Diseases Research), National Health Commission Key Laboratory of Parasite and Vector Biology, WHO Collaborating Center for Tropical Diseases, National Center for International Research on Tropical Diseases, Shanghai, China.
PLoS Negl Trop Dis. 2025 Apr 8;19(4):e0013007. doi: 10.1371/journal.pntd.0013007. eCollection 2025 Apr.
Necator americanus is the predominant species causing hookworm infections in humans. Despite advancements in prevention strategies, mild cases of infection still occur, highlighting the need for improved detection technology. Recombinase Polymerase Amplification (RPA) is an isothermal molecular diagnostic known for its sensitivity, speed, portability, and widespread application in detecting various pathogens. Although several molecular assays are available for N. americanus, they have limitations in detecting mild N. americanus infections.
Fluorescent RPA primers and probes targeting the N. americanus internal transcribed spacer 2 (ITS2) gene were developed. The method's detection limit was assessed via serial dilution of genomic DNA. Specificity was confirmed against Clonorchis sinensis, Schistosoma japonicum, Fasciola hepatica, Ascaris lumbricoides, Enterobius vermicularis and Ancylostoma duodenale. Thirty samples identified as positive by Kato-Katz, along with 11 samples identified as negative by the method, were tested to evaluate the sensitivity and specificity of fluorescent RPA. Additionally, 287 field samples were tested for validation with these methods. All positive samples were identified as either N. americanus or A. duodenale.
This study successfully developed a fluorescent RPA assay targeting the ITS2 gene of N. americanus. The length of the amplified fragment was 237 bp. Optimized conditions were achieved, resulting in a minimum detection limit of 1fg/µL, with no cross-reactivity with other pathogens. In laboratory validation, the fluorescent RPA assay demonstrated 100% sensitivity (30/30) and 100% specificity (11/11) compared to the Kato-Katz, and 100% sensitivity (29/29) and 91.7% specificity (11/12) when compared to the semi-nested PCR. In field validation using human fecal samples, the fluorescent RPA assay showed a sensitivity of 90.0% (36/40) and a specificity of 91.1% (225/247) compared to the Kato-Katz. And the sensitivity of the fluorescent RPA method compared to the semi-nested PCR method was 100% (34/34), while the specificity was 90.5% (229/252).
The fluorescent RPA assay presents a rapid and dependable method for detecting N. americanus in fecal samples. Its high sensitivity and specificity provide significant utility for field surveillance and early identification of N. americanus infections. This advancement could facilitate the rapid molecular diagnosis of N. americanus disease in hookworm-endemic regions.
美洲板口线虫是导致人类钩虫感染的主要虫种。尽管预防策略有所进步，但轻度感染病例仍有发生，这凸显了改进检测技术的必要性。重组酶聚合酶扩增技术（RPA）是一种等温分子诊断方法，以其灵敏度高、速度快、便于携带以及在检测各种病原体方面的广泛应用而闻名。虽然有几种分子检测方法可用于检测美洲板口线虫，但它们在检测轻度美洲板口线虫感染方面存在局限性。
开发了针对美洲板口线虫内部转录间隔区2（ITS2）基因的荧光RPA引物和探针。通过基因组DNA的系列稀释评估该方法的检测限。针对华支睾吸虫、日本血吸虫、肝片吸虫、蛔虫、蛲虫和十二指肠钩口线虫确认了特异性。对30份经加藤厚涂片法鉴定为阳性的样本以及11份经该方法鉴定为阴性的样本进行检测，以评估荧光RPA的灵敏度和特异性。此外，对287份现场样本使用这些方法进行检测以进行验证。所有阳性样本均被鉴定为美洲板口线虫或十二指肠钩口线虫。
本研究成功开发了一种针对美洲板口线虫ITS2基因的荧光RPA检测方法。扩增片段长度为237bp。实现了优化条件，最低检测限为1fg/µL，且与其他病原体无交叉反应。在实验室验证中，与加藤厚涂片法相比，荧光RPA检测方法的灵敏度为100%（30/30），特异性为100%（11/11）；与半巢式PCR相比，灵敏度为100%（29/29），特异性为91.7%（11/12）。在使用人类粪便样本的现场验证中，与加藤厚涂片法相比，荧光RPA检测方法的灵敏度为90.0%（36/40），特异性为91.1%（225/247）。与半巢式PCR方法相比，荧光RPA方法的灵敏度为100%（34/34），特异性为90.5%（229/252）。
荧光RPA检测方法为检测粪便样本中的美洲板口线虫提供了一种快速可靠的方法。其高灵敏度和特异性为美洲板口线虫感染的现场监测和早期识别提供了重要作用。这一进展有助于在钩虫流行地区快速进行美洲板口线虫病的分子诊断。
