Study On The Molecular Detection Techniques Of Bovine Neosporosis

Neosporosis is a kind of protozoal disease caused by Neospora caninum parasitizes in various kinds of livestock such as dog, cattle, horse and sheep. It is primarily causative agent of abortion, stillbirth and nervous system disease of nascent calves in cattle and has brought about huge economic loss to the livestock husbandry. Currently, there is no effective drug and vaccines to control neosporosis, so it is necessary to develop an early, accurate diagnostic method for controlling neosporosis. In our study, we made use of the achievement of former people, the rapid and accurate methods for detecting the bovine neosporosis were established using three PCR methods (two-temperature PCR, real-time fluorescent PCR, real-time fluorescent PCR with Internal Amplification Control) and gene chip. The results are as follows:1. A two-temperature PCR method was established to detect the bovine neosporosis according to the method of uniform design, the standard of neosporosis was to gain the special Nc-5 gene core fragment of Neospora caninum. The results showed that a 138 bp target fragment was amplified successfully. Recombined plasmid was digested by BamH I and Hind III and amplified by PCR, both gained the expected fragment. The acquired sequence of Nc-5 gene fragment shared 98.55% homology with the Nc-5 gene sequence of N. caninum(X84238.1) in GenBank.2. A real-time fluorescent PCR assay to detect bovine neosporosis was established. The results showed that the developed method was a specific and sensitive, the sensitive limit may reach 10 gene copies nucleic acid molecule per PCR reaction, which was 1000 fold higher than that of conventional PCR, the correlation coefficient of the standard curve was up to 0.993 with the Nc-5 plasmid and the assay could be performed in 2 hour or less.3. The Internal Amplification Control(IAC) template of real-time fluorescent PCR was achieved by designing primers and amplifying with bridge-building PCR method. The detection limit was about 10 copies nucleic acid molecule per PCR reaction. The real-time fluorescent PCR with IAC was 1000 fold than that of conventional PCR. This result was almostly coincident with the conventional real-time PCR without IAC. It suggested that this method was useful in the rapid detection of bovine neosporosis and the lab quality control for indicating and proofreading the false negative results by real time detecting the full reaction.4. According to the published Nc-5 gene sequence of Neospora caninum(N. caninum) and the gB gene sequence of Bovine Herpesvirus 1(BHV-1) in GenBank, high conservative primers and two oligonucleotide probes were designed. Following purification, the metamorphic labeled PCR products were hybridized to the gene chip, fluorescence signals were scanned and analyzed, and then the gene chip diagnostic technique was established. The gene chip was proved to be a specific, sensitive, reliable and simple method and could be used to detect the two pathogeny from clinical sample rapidly.This study successfully established three PCR methods and gene chip to detect bovine neosporosis, and solved the difficult problem for bovine neosporosis in being, with afford application value in the rapid detection of bovine neosporosis .