Development and evaluation of PCR assays for the detection of Paenibacillus larvae in honey samples:: Comparison with isolation and biochemical characterization
Development and evaluation of PCR assays for the detection of Paenibacillus larvae in honey samples:: Comparison with isolation and biochemical characterization
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DOI：
10.1128/aem.69.3.1504-1510.2003
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发表时间：
2003-03-01
影响因子：
4.4
通讯作者：
Nowotny, N
中科院分区：
文献类型：
--
作者：
Bakonyi, T;Derakhshifar, I;Nowotny, N
PCR 测定法用于直接检测蜂蜜样品中的类芽孢杆菌幼虫，并与分离和生化表征程序进行比较。对从 16S rRNA 和金属蛋白酶前体基因区域设计的不同引物对以及不同的 DNA 提取方法进行了测试和比较。通过连续稀释从 P. larvae 培养物中获得的 DNA 提取物来评估反应的灵敏度。通过分析从蜂蜜中分离的相关类芽孢杆菌和芽孢杆菌菌株来评估引物的特异性。 PCR 检测也扩增了这些相关细菌，但灵敏度较低。下一步，PCR 检测适用于来自 15 个国家的受污染蜂蜜和其他蜂产品。溶菌酶处理后进行蛋白酶 K 消化被确定为 P. larvae 孢子的最佳 DNA 提取方法。最敏感的引物对在 23 个受污染的蜂蜜样品中的 18 个以及花粉、蜡和蜂巢中检测到了 P. 幼虫。含有腐生杆菌和类芽孢杆菌（但不含腐生杆菌幼虫）的蜂蜜样本 PCR 呈阴性。尽管分离和生化鉴定方法 (BioLog) 显示出较高的灵敏度和特异性，但 PCR 被证明是大规模筛选美国腐臭幼虫蜂蜜样品的一种有价值的技术，特别是考虑到其快速性和适中的成本。
PCR assays were developed for the direct detection of Paenibacillus larvae in honey samples and compared with isolation and biochemical characterization procedures. Different primer pairs, designed from the 16S rRNA and the metalloproteinase precursor gene regions, and different DNA extraction methods were tested and compared. The sensitivity of the reactions was evaluated by serial dilutions of DNA extracts obtained from P. larvae cultures. The specificity of the primers was assessed by analyzing related Paenibacillus and Bacillus strains isolated from honey. The PCR assays also amplified these related bacteria, but at lower sensitivity. In the next step, the PCR assays were applied to contaminated honey and other bee products originating from 15 countries. Lysozyme treatment followed by proteinase K digestion was determined to be the best DNA extraction method for P. larvae spores. The most sensitive primer pair detected P. larvae in 18 of 23 contaminated honey samples, as well as in pollen, wax, and brood. Honey specimens containing saprophyte bacilli and paenibacilli, but not P. larvae, were PCR negative. Although the isolation and biochemical identification method (BioLog) showed higher sensitivity and specificity, PCR proved to be a valuable technique for large-scale screening of honey samples for American foulbrood, especially considering its rapidity and moderate costs.