Improved detection of endoparasite DNA in soil sample PCR by the use of anti-inhibitory substances
Improved detection of endoparasite DNA in soil sample PCR by the use of anti-inhibitory substances
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DOI：
10.1016/s0304-4017(02)00199-1
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发表时间：
2002-09-26
影响因子：
2.6
通讯作者：
Epe, C
中科院分区：
文献类型：
--
作者：
Krämer, F;Vollrath, T;Epe, C
虽然已经有许多微生物检查土壤中是否存在人类致病性发育寄生虫阶段的犬钩虫和犬弓蛔虫，但分子技术（如DNA提取，纯化和随后的PCR）几乎没有应用。在这里，用PCR检测了人工污染基因组DNA或寄生虫卵的土壤样品的DNA制备物。A. caninum和T.基于ITS-2序列的犬特异性引物用于扩增。在纯DNA制备后，仍可检测到高含量的PCR干扰物质。随后，使用两种不同的PCR干扰剂抑制剂（GeneReleaserTM，Bioventures Inc.和Maximator（（R）），Connex GmbH）在PCR中进行比较。这两种物质都大大提高了PCR的灵敏度。然而，比较用两种化合物实现的灵敏度增加证明了Maximator（（R））的优越性，其将灵敏度提高到允许阳性检测单个A的程度。犬卵和3个T.土壤样本中的狗卵这种程度的敏感性不能达到与基因释放（TM）的任何寄生虫此外，最大化（R）不仅增加了敏感性，它也成本较低，需要较少的时间，并具有较低的污染风险。讨论/阐述了分子方法在土壤样品流行病学检查中的未来应用。(C)2002 Elsevier Science B. V.保留所有权利。
Although there have been numerous microbial examinations of soil for the presence of human pathogenic developmental parasite stages of Ancylostoma caninum and Toxocara canis, molecular techniques (e.g. DNA extraction, purification and subsequent PCR) have scarcely been applied. Here, DNA preparations of soil samples artificially contaminated with genomic DNA or parasite eggs were examined by PCR. A. caninum and T. canis-specific primers based on the ITS-2 sequence were used for amplification. After the sheer DNA preparation a high content of PCR-interfering substances was still detectable. Subsequently, two different inhibitors of PCR-interfering agents (GeneReleaser(TM), Bioventures Inc. and Maximator((R)), Connex GmbH) were compared in PCR. Both substances increased PCR sensitivity greatly. However, comparison of the increase in sensitivity achieved with the two compounds demonstrated the superiority of Maximator((R)), which enhanced sensitivity to the point of permitting positive detection of a single A. caninum egg and three T. canis eggs in a soil sample. This degree of sensitivity could not be achieved with GeneReleaser(TM) for either parasite Furthermore, Maximator((R)) not only increased sensitivity; it also cost less, required less time and had a lower risk of contamination. Future applications of molecular methods in epidemiological examinations of soil samples are discussed/elaborated. (C) 2002 Elsevier Science B.V. All rights reserved.