Discrimination of gastrointestinal nematode eggs from crude fecal egg preparations by inhibitor-resistant conventional and real-time PCR.
Discrimination of gastrointestinal nematode eggs from crude fecal egg preparations by inhibitor-resistant conventional and real-time PCR.
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DOI：
10.1371/journal.pone.0061285
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Krücken J
中科院分区：
文献类型：
--
作者：
Demeler J;Ramünke S;Wolken S;Ianiello D;Rinaldi L;Gahutu JB;Cringoli G;von Samson-Himmelstjerna G;Krücken J
胃肠道线虫的诊断主要依赖于辅镜检查方法，如浮选法、加藤卡茨法、麦克马斯特法或FLOTAC法。尽管FLOTAC可以进行准确的定量，但许多线虫卵只能区分为属或科。一些区分密切相关物种的分子诊断工具存在从粪便中分离DNA的成本高和灵敏度有限的问题，因为大多数试剂盒只使用少量的粪便(1克)。为了与FLOTAC完全兼容，设计了一种直接从粗蛋制剂中进行聚合酶链式反应的方法，以准确地定量每克粪便中的鸡蛋数(EPG)并确定种类组成。从浮选液中回收鸡蛋，并通过筛分进行浓缩。裂解是通过反复煮沸和冷冻循环实现的-只有鞭虫卵需要额外的机械破坏。直接以鸡蛋裂解产物为模板，用对PCR抑制剂具有特别抗性的Phusion DNA聚合酶进行PCR。从山羊、牛、马、猪、猫、狗和小鼠的粪便中获得定性结果。最终建立的方法也适用于在EvaGreen存在下的实时定量聚合酶链式反应，当提取液稀释至少四倍时，没有检测到聚合酶链式反应抑制。灵敏度与DNA分离方法相当，可以可靠地检测到添加了5个EPG的样品。对于猫弓形虫，检测下限低于一个EPG。高分辨率熔融(HRM)分析是区分猫弓形虫和犬弓形虫的可能，是一种快速鉴定物种的工具。采用限制性片段长度多态性(RFLP)和HRM分析方法对美国钩虫和十二指肠钩口线虫进行了基因分型。该方法通过提高速度和灵敏度，同时降低总体成本，能够显著提高胃肠道线虫的分子诊断水平。为了鉴定菌种或抗性等位基因，可以使用RFLP、反向线条杂交、Sanger测序和HRM等多种不同的后处理方法对PCR产物进行分析。
Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g). A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles – only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post PCR methods can be used such as RFLP, reverse-line-blot, Sanger sequencing and HRM.
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影响因子：
2.2
作者：
Costa, Jean-Marc;Cabaret, Odile;Bretagne, Stephane
通讯作者：
Bretagne, Stephane
影响因子：
3.2
作者：
Leles D;Gardner SL;Reinhard K;Iñiguez A;Araujo A
通讯作者：
Araujo A
比较Kato-Katz，Koga琼脂板，以太浓度和FLOTAC的诊断精度，用于曼森和土壤传播的蠕虫。
影响因子：
3.8
作者：
Glinz, Dominik;Silue, Kigbafori D.;Knopp, Stefanie;Lohourignon, Laurent K.;Yao, Kouassi P.;Steinmann, Peter;Rinaldi, Laura;Cringoli, Giuseppe;N'Goran, Eliezer K.;Utzinger, Juerg
通讯作者：
Utzinger, Juerg
影响因子：
2.6
作者：
Ionita, Mariana;Howe, Daniel K.;Yeargan, Michelle
通讯作者：
Yeargan, Michelle
影响因子：
1.9
作者：
Habtamu, Kassahun;Degarege, Abraham;Erko, Berhanu
通讯作者：
Erko, Berhanu