Application Research On The Detection Of Hookworm Eggs In Human Faecal Samples With PCR Based On ITS Gene

【Objective】Recently, Hookworm infections are still endemic in low and middle income tropical and subtropical countries with extensive impact on the socioeconomic and public health. Ancylostomiasis is still an unsolved public health problem in many rural and remote areas of the world with economic poverty and lagging development.Currently microscopy used to examination and identify hookworm ova to the species level are time consuming and lack accuracy. So,there is a great practical value to develop a accurate and rapid method of precise detection and differentiation of species involved. The target of the study is developing a method of accurate identification of hookworm species involved by the semi-nested polymerase chain reaction(PCR) through extracting DNA from hookworm ovum in human fecal samples. Effect of semi-nested PCR for detecting hookworm was evaluated by comparing the results between PCR and microscopy.【Methods】(1) Faecal sample collection. Faecal samples from humans were collected from the fifth-grade students of various primary schools in Jinghong City and Menghai County of Xishuangbanna Prefecture and from the third to fifth grade students in Jiangcheng Country of Pu’er City. All faecal samples were detected thrice by utilizing the Kato-Katz method microscopically to identify the parasite ovum of infection species and count them independently.66 stool samples containing hookworm ovum were selected, some of them accompany with the infection of roundworm, whipworm or tapeworm.20 excrement samples without hookworm ovum were sorted out randomly as negative control, some of them include roundworm andwhipworm ovum. Samples were deposited in labeled 5ml cryogenic vials with 95%ethanol and refrigerated at 4℃ until further analysis.( 2) Application of PCR technique :A semi-nested PCR method was used to amplify the second internal transcribed spacer(ITS2) and 28 S RNA region of ribosomal DNA of hookworm ovum in all collected feces samples for detection and identification. The PCR product was run on a 2% agarose gel, some of PCR-positive amplicons were subjected to DNA sequencing in both directions. The nucleotide sequences were analyzed by BLAST(nucleotide blast) to identify hookworm species.At the same time,the methods of DNA extraction was improved and elimination of nonspecific amplication in nested PCR was seeked. Two-fold dilution method was used to detect the sensibility of semi-nested PCR.【Results】After improvement of DNA extraction process,66 faecal samples of hookworm egg positive identified by microscopy, sensitivity of PCR of 61.12%(41of 66)improved to 96.97%(64 of 66). The study found that N.americanus 98.4%(63 of64)was the most predominant hookworm species infecting humans in southern Yunnan, followed by A.ceylanicum1.56%(1 of 64),no A.duodenale was found. No other specific fragment was amplified, specificity of the semi-nested PCR is 100%,consistency rate is 97.67%, illustrating that the two methods are of good consistency.Sensibility test results showed that the semi-nested PCR lower limit of detection was the stock solution diluted to 256-fold(approximately 4 hookworm eggs per gram feces).【Conclusion】1. The results indicated that the semi-nested PCR is a accurate, rapid, sensitive and specific approach after improvement, it is a tool has practical value for detection and identification of hookworm ova in human faeces samples in epidemiological survey.2. The improvement of trituration is conducive to broke the egg-shells and egg membranes of hookworm eggs and to improve the success rate of DNA extraction of hookworm eggs.3.Reducing concentration of outer primer can eliminate the non-specific amplification of the semi-nested PCR.4.Discovery of infection of A. ceylanicum prompt that the hookworm disease have risk of mutual infection between human and animals.