Classification Method Of PCR Detection With Two Species Of Hookworm Eggs In Human Excrement

Objective: To explore a new method for classification and identification ofAncylostoma duodenale and Necator americanus: using PCR technique amplifiesCOI gene fragment of the hookworm eggs from human fecal samples.Methods:The experiment is divided into two parts, field sampling and laboratorytesting. The field work is collecting stool samples from the fourth grade studentsin Jinghong city and Menghai County of Xishuangbanna Prefecture. TheKato-Katz technique is used to examine the stool samples three times by themicroscopic examination to determine the types of parasite eggs and count all kindsof eggs density respectively. Collect stool samples including positive specimenscontaining hookworm eggs, as well as the negative samples without hookwormeggs; Thses samples are placed in the95%ethanol at4refrigerator for the furtherexperiments. The PCR method is used to amplify COI gene fragment ofthe hookworm eggs. The PCR products was sequenced by the Gene Company. Thesequence was align by BLAST in NCBI to determine gene types of hookworm eggs.Results:40fecal samples of the hookworm eggs was obtained, which the26samples contain only the hookworm egg samples, and in addition to3samplescontain Ascaris eggs and5samples contain Trichuris trichiura and6samples contain the hookworm eggs, Ascaris eggs,whipworm eggs. Select10doesnot contain the eggs of hookworm or does not contain any eggs in stool samples wereused as negative control samples. After PCR amplification, the PCR product was runagarose gel electrophoresis to show positive or negative results.14microscopypositive samples appeared positive bands, position between300bp-500bp. thesensitivity is35%(14/40). After sequencing BLAST comparison, a similar rate of6samples with Necator americanus COI gene is in84%-99%. The false positive rate is57%(8/14). The10negative specimens were amplified by PCR, agarose gelelectrophoresis were no band. The specificity is100%.Conclusion： Experiments show that the amplification of DNA extracted fromhookworm eggs in feces by PCR technology to identify the hookworm infection typeis feasible, but because the preliminary experimental detection rate is low, furtherresearch needs to improve experimental methods, the sensitivity, specificity and so on.