The Application Of Nested PCR Technique To Identify Human Hookworm From Excrement Eggs

Objective: This paper aims at exploring a new method of classifying and identifying the major human parasitic hookworms by the nested PCR technique through extracting the DNA of hookworm eggs from human stool samples. Contents and Methods: There are two parts in the experiment: field sampling and laboratory testing. The task of field sampling is collecting stool samples of fourth grader from more than one school in Jinghong city, xishuangbanna Dai Autonomous Prefecture, Yunnan province and Menghai county. Each stool sample will be tested for three times by using Kato Katz method, then with the help of microscopic examination to determine the type of parasite eggs and have them counted, select 39 stool samples with hookworm eggs that have been microscopic examined as the positive sample and 21 stool samples without hookworm eggs( part of them contains the roundworm) as the negative control samples, then place the samples in the 95% ethanol at 4C refrigerator for next detection. In the lab, the internal transcribed spacer 2(ITS-2) target gene will be amplified by using the nested PCR technique as well as sequence the target gene, and then BLAST comparison will be carried out to determine the gene type.Results: After nested PCR amplification and the BLAST comparison,its DNA sequence is quite similar to that of the American hookworm’s with the rate of 99%, and with one sample’s similar rate at 99% to the Ceylon hookworm ribosomal gene, which are identified as Necator americanus and Ancylostoma. Its detection sensitivity is 92.31%(36/39); specificity is 33.33%(7/21). After the results of microscopic examination and the nested PCR technique by X2 test, X2=4.747,P=0.029. The difference between these two detection methods has statistical significance, but the detection rate of the nested PCR(50/ 60 is higher than that of microscopic examination(39/60). Conclusion: In this study, an experimental method is established by using the nested PCR technique to identify and detect hookworm in human stool and proves that its detection rate is higher than that of Kato Katz, which lays a preliminary foundation for achieving the target of discriminating and diagnosing a variety of hookworms as fast as possible. This study for the first time identified the case of infecting ceylanicum hookworm(Ancylostoma) in Yunnan province, which indicates that both human beings and animals are at the risk of getting infected with the Yunnan hookworm in Yunnan province. Therefore, it is worthwhile to conduct further study in the future.