Gao Pengya, Wu Changde, Zhang Jin, Wang Shuping, Huang Ying, Dong Yinping, Liu Tingting, Ye Changyun, Xu Xuefang, Xin Wenwen
State Key Laboratory for Infectious Disease Prevention and Control and National Institute for Communicable Diseases Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China.
Front Microbiol. 2022 May 9;13:860992. doi: 10.3389/fmicb.2022.860992. eCollection 2022.
is the causative pathogen of botulism. Laboratory detection of is essential for clinical therapy treatment of botulism due to the difficulty in diagnosis, especially in infant botulism. The extreme toxicity of botulinum neurotoxin (BoNT) requires a sensitive detection method. Due to the detection limit of real-time quantitative PCR (q-PCR), a more sensitive detection method, micro-drop digital PCR (ddPCR) was applied in main serotypes A and B. The following performance criteria were evaluated by ddPCR: analytical sensitivity; repeatability; and diagnostic specificity. The limit of detection (LOD) was 0.84 and 0.88 copies/μl for BoNT A and B genes, respectively, by ddPCR with high specificity, compared to 5.04×10 and 6.91×10 copies/μl by q-PCR. It was increased 10 times compared with q-PCR in spiked stool samples. This improvement in sensitivity was especially important in clinical samples as more positive samples were detected by digital PCR compared with q-PCR. Meanwhile, enrichment time for low bacteria content samples was shortened by four hours both in serotypes A and B by ddPCR compared with q-PCR, which are important for laboratory diagnosis and epidemiology work.
是肉毒中毒的致病病原体。由于肉毒中毒诊断困难，尤其是婴儿肉毒中毒，因此对其进行实验室检测对于临床治疗至关重要。肉毒杆菌神经毒素（BoNT）的极高毒性需要一种灵敏的检测方法。由于实时定量PCR（q-PCR）的检测限，一种更灵敏的检测方法——微滴数字PCR（ddPCR）被应用于主要血清型A和B。通过ddPCR评估了以下性能标准：分析灵敏度、重复性和诊断特异性。与q-PCR分别为5.04×10和6.91×10拷贝/微升相比，ddPCR对BoNT A和B基因的检测限分别为0.84和0.88拷贝/微升，特异性高。在加标的粪便样本中，与q-PCR相比，灵敏度提高了10倍。这种灵敏度的提高在临床样本中尤为重要，因为与q-PCR相比，数字PCR检测到更多阳性样本。同时，与q-PCR相比，ddPCR使血清型A和B中低细菌含量样本的富集时间均缩短了4小时，这对实验室诊断和流行病学工作很重要。
