Establishment And Application Of Loop-Mediated Isothermal Amplification For Detetion Of Giardia SPP.

Giardia is a genus of intestinal parasitic protozoa that infects a wide range of vertebrate hosts. The giardiasis caused by Giardia spp. distributed worldwide. Since the 1970s, many cases caused by Giardia lamblia were reported around the world. For the reason of popular among the international tourists, this disease was called as "traveler’s diarrhea". Now, it was been listed as on of the 10 main parasitic diseases of human. With the development of the society, the relationship between human and animals became more and more close, with the increasing of the direct or indirect contact with animals, more and more risks and potential threat to human health would occur.Currently, the methods used to detect Giardia Spp. were based on microscopic examination or PCR amplification. In microscopic examination, oocyts in the samples used to be missed detecting by the checker. To perform PCR detection, specific instrument should be provided. To establish peculiar, efficient and low cost method to detect Giardia spp., loop-mediated isothermal amplification (LAMP) technology was established and applied in fecaes sample detection.1. First of all, Giardia lamblia was identified by morphological observation and PCR detecting. The triose phosphate isomerase (TPI) and the β-giardin gene were amplified by PCR, and the fragments were cloned into the pMD-19T vecter. The recombinant plasmid named as pMD-19T-TPI and pMD-19T-β-giardin, were used as the templates for the establishment of LAMP detection.2. Primeres for LAMP and the PCR amplification were designed based on the TPI and the β-giardin genes of Giardia lamblia. To test the sensitivity of the LAMPs, pMD-19T-TPI and pMD-19T-β-giardin were diluted to different concentrations and used as templates. Then the LAMP and PCR were performed. Results showed that the minima concentrations of DNA that LAMP could be detected were 1.85×10-9ng and 1.65×10-9ng. PCR’s resulting showed that the minima concentrations of DNA were 7.4×10-7ng and 6.6×10-7ng. The sensitivity of the LAMP method was 400 times than that of PCR. To check the specificity of the LAMP, genomes from Giardia lamblia, mixed Eimeria (Eimeria tenella and Toxic Eimeria), Trichinella spiralis, Toxoplasma gondii, Escherichia coli and Streptococcus suis were used as the templates, and then LAMPs wereperformed. Only the reaction added Giardia lamblia genome as the template showed fluorescent green after adding SYBR Green I. DNA ladder was observed by agrose gel electrophoresis. The results indicated that this LAMP method had high specificity.3.69 fecal samples from dogs and wild animals were collected and analyzed with microscopic examination, PCR technique and LAMP technique.8 samples (11.6%) were found Giardia positive by using microscopic examination. Meanwhile,13 samples (18.8%) were detected positive with PCR and LAMP. In this study, PCR and LAMP showed higher sensitivity than the ordinary microscopic examination.Low costs, high performance (sensitivity and specificity) and handy for operation were the advantages of LAMP method, which means this method could provide a better choice in diagnosing giardiasis.