Study On Nested Pcr For The Microdetection Of Cryptosporidium Oocysts And Its Application

Cryptosporidium oocysts were separated from the faeces in a cattle farm and identified as Cryptosporidium muris according to their morphology, structure arid size after a sheather's floating and an auramine-phenol and acid-fast-staining (AP-AFS).Two sets of special primers to Cryptosporidium and Cryptosporidium muris were designed and synthesized respectively based on the sequence of 18s rDNA. A special fragment of 540bp was amplified after polymerase chain reaction (PCR) with a series of suitable reaction parameters and in an optimal concentration of Mg2+ and Taq polymerase. Then the products of primary PCR were used as templates, oligonucleotides special to Cryptosporidium muris as primers in the Nested PCR, a fragment of 250 bp was amplified, while no fragment was observed in the testing of other samples from disease pathogens such as Eimeria coccidian, Typansooma Evansi, giardias, E.col et al.A minimal number from 1 to 10 of oocysts could be detected by the Nested PCR. Compared with other methods, the Nested PCR might be provided with the highest sensitivity for the detection, whose sensitivity was 103 to 10s times higher than that of conventional PCR, sheather's floating and AP-AFS.In order to enhance the practicability of the Nested PCR, four methods of extracting DNA from oocysts of Cryptosporidium muris were tested and evaluated. It was found that the following easier treatment of oocysts appeared higher sensitive: the oocysts were suspended in 2% DTT, fractured by 3 cycles of freezing in liquid nitrogen and thawing in room temperature or 8 cycles freezing at -20癈 and thawing at room temperature, then centrifuged at 13 OOOg.In addition, the Nested PCR was used in examining 119 of clinic samples, a positive rate of 23.29%, 11.11% and 0.00% for Cryptosporidium spp. respectively in faeces, milk and polluted water was gained, that showed its sensitivity was higher than that of conventional PCR, sheather's floating and AP-AFS.Briefly, the new Nested PCR has a bright prospect of application in the microdetection of Cryptosporidium oocysts because of its high specificity and sensitivity.