Masny Aleksander, Rozej Wioletta, Gołab Elzbieta
Narodowy Instytut Zdrowia Publicznego-Państwowy Zakład Higieny Zakład Parazytologii Lekarskiej.
Med Dosw Mikrobiol. 2009;61(3):259-65.
PCR detection of genetic material of the parasites present in faeces may be an alternative for microscopic and serological tests routinely used for diagnosing parasitic enteral infections. However, small amount of target DNA combined with low efficiency of total DNA extraction, and presence of PCR inhibitors in the samples to be amplified, may cause false negative detection results. The aim of this work was to evaluate the impact of DNA isolation procedure used on the amplification of DNA fragments from the genomes of protozoan Cryptosporidium parvum and the nematode Trichinella spiralis. Two methods based on different principles of biological material lysis were evaluated; NucliSENS miniMAG employing simultaneously applied chemical lysis and mechanical disruption or mechanical disruption followed by enzymatic lysis in case of QIAamp DNA Stool Mini Kit. Both of the analyzed systems for nucleic acids purification allowed isolation of DNA from purified Cryptosporidium parvum oocysts and Trichinella spiralis muscle larva mixed with mouse or pig faeces. However, sensitivity of PCR detection of DNA obtained by enzymatic lysis based method was lower compared to the one achieved with DNA isolated by chemical lysis. When QIAamp DNA Stool Mini Kit procedure was used detection limit was 5 and 10 fold higher for Cryptosporidium parvum and Trichinella spiralis, respectively. Only NucliSENS miniMAG procedure combined with mechanical disruption of the faecal material allowed detection of Cryptosporidium sp. in human fecal samples collected from children with diahorrea, and detection of Trichinella spiralis in stool samples obtained, on days 8 to 12 post infection, from mice experimentally infected with 500 muscle larvae per mouse. Thorough mechanical disruption with simultaneous chemical lysis allows efficient isolation ofDNA of the investigated intestinal parasites present in stool and the subsequent PCR detection.
通过聚合酶链反应（PCR）检测粪便中寄生虫的遗传物质，可能是常规用于诊断寄生虫肠道感染的显微镜检查和血清学检测的替代方法。然而，目标DNA量少，加上总DNA提取效率低，以及待扩增样品中存在PCR抑制剂，可能会导致假阴性检测结果。这项工作的目的是评估所使用的DNA分离程序对原生动物微小隐孢子虫和线虫旋毛虫基因组DNA片段扩增的影响。评估了基于生物材料裂解不同原理的两种方法；NucliSENS miniMAG采用同时应用化学裂解和机械破碎，而对于QIAamp DNA Stool Mini试剂盒，则先进行机械破碎，然后进行酶解。两种分析的核酸纯化系统都能从纯化的微小隐孢子虫卵囊和与小鼠或猪粪便混合的旋毛虫肌肉幼虫中分离出DNA。然而，与化学裂解分离的DNA相比，基于酶解方法获得的DNA的PCR检测灵敏度较低。当使用QIAamp DNA Stool Mini试剂盒程序时，微小隐孢子虫和旋毛虫的检测限分别高出5倍和10倍。只有NucliSENS miniMAG程序与粪便材料的机械破碎相结合，才能在从腹泻儿童收集的人类粪便样本中检测到隐孢子虫属，并在感染后第8至12天从每只小鼠实验感染500条肌肉幼虫的小鼠粪便样本中检测到旋毛虫。彻底的机械破碎与同时进行的化学裂解能够有效地分离粪便中所研究的肠道寄生虫的DNA，并随后进行PCR检测。
