| Porcine respiratory disease complex(PRDC)is a common disease characterized by dyspnea,cough, fever and growth retardation, which is caused by a combination of many factors, such as viruses, bacteria, mycoplasma, parasites and environmental stress. According to the Etiology analysis, Porcine circovirus type 2(PCV-2), Porcine reproductive and respiratory syndrome virus(PRRSV), Mycoplasma hyopneumoniae(Mhp), Actinobacillus pleuropneumoniae(APP),Haemophilus parasuis(HPS), Classical swine fever virus(CSFV), Porcine parvovirus(PPV)and Swine influenza virus(SIV) were the main pathogens. Among them, APP, HPS, Mhp, PCV-2,PRRSV, and CSFV are common in our country.The muti-pathogen mixed infection or secondary infection significantly increases difficulty in the clinical diagnosis. However, at present, there are some diagnosis methods detecting the single one, such as separation of culture, serological detection and PCR amplification, lacking of a way to detect the six pathogens simultaneously. Therefore, the establishment of this special methods could save the test cost and improve the detection rate,contributing to the epidemiological investigation,disease surveillance and entry and exit inspection and quarantine.In this study, we selected APP, HPS, Mhp, PCV-2, PRRSV and CSFV as the research objects, anddeveloped an oligonucleotide microarray detection of six pathogens, which possessed more specific and stable characteristics. The main contents and results are as follows.1. Design primers and probes.Based on the sequence of APP, HPS, Mhp, PCV-2, PRRSV and CSFV, we designed 6 pairs of specific primers and a plurality of probes with the software of Mega5, Primer Premier 5.0 and Oligo6.0, and amplified nucleic acid of six pathogens by PCR and constructed T-cloning vectors.2. Probe screening and the establishment of a single pathogenic gene chip detection method.Referenced the marking method of GeXP, we used Cy3 labeling target sequence and hybridized with probes that was spotted on the aldehyde slide, then scanned the result of hybridization and screened the specific probes. On the basis of the selection of the probe, we optimized annealingtemperature, outercycles, the concentration of hybridization probe, hybridization solution, and hybridization temperature and time. The optimized reaction conditions followed as annealing temperature was 56~58℃, outer loop 30 times of amplification, and the PCR products mixed with hybridization solution was protected from avoided light and hybridized for 3h at 42℃. Depended on the specificity test, sensitivity test and stability test to evaluate detection method, the results indicated that the method was specific and stable. What’s more, the detection limits of oligonucleotide microarray were 2.97×103copies/μL for APP, 4.38×103copies/μL for HPS,8.26×102copies/μL for Mhp, 3.78×102copies/μL for PCV-2, 1.38×103copies/μL for PRRSV and3.32×10copies/μL for CSFV, respectively. Besides, Compared with ordinary PCR, the sensitivity of oligonucleotide microarray was 10~100 times more than the common one.3. The establishment and assessment of oligonucleotide microarray for multi-pathogen.On the basis of single pathogen detection method, optimizing the concentration of six pairs of specific chimeric primers through orthogonal experiment, and preliminary establishing an oligonucleotide microarray to detect six kinds of pathogens synchronously. We optimimzed the hybridization temperature and time, then evaluated the method.Specificity test: we conducted the amplification and hybridization of Pseudo rabies virus,Porcine parvovirus, Japanese B encephalitis virus, Swine vesicular disease virus, Vesicular stomatitis virus, Foot-and-mouth disease virus, Bluetongue virus, Peste des petits ruminants virus and Salmonella, and verified the specificity of microarray.The results suggested that there was no cross-reaction with mentioned pathogens.Sensitivity test: we tested with the mixed six positive plasmids as a template which was diluted proportionally..The results manifested that the detection limits of oligonucleotide microarray were5.8×102copies/μL for APP, 7.8×103copies/μL for HPS, 6.8×103copies/μL for Mhp, 6.3×102copies/μL for PCV-2, 4.8×103copies/μL for PRRSV and 5.5×102copies/μL for CSFV, respectively.Therefore,the detection limits of oligonucleotide microarray was 103copies/μL through synthetical consideration.Repeatability test: we selected six substrates of the same batch and six substrates of different batches to do intra-assay and inter-assay test. The results showed that the intra-assay and inter-assay coefficients of variation were smaller than 5.0%.Preserve time test: the microarray was preserved for four months and we groped the optimum storage temperature and time of the chip. The results elucidated that the preserve timeof gene chip was four months at-20℃.Preliminary clinical application:we detected the 186 nasal swabs samples in ChongQing using the gene chip and standard method, respectively. The results indicated that the single infection was34 and mixed infection was 11 by gene chip, and the single infection was 28 and the mixed infection was 13 by the standard method. Subsequently, the positive samples were sent to sequence, the results and the detection rate of oligonucleotide microarray and sequence were identical, whichsuggested the oligonucleotide microarray apply to the clinical test of muti-pahtogen.In this study, we have established the oligonucleotide microarray, which provides a rapid and high-throughput method for six pathogenic epidemiology investigation and clinical diagnosis, and lays the foundation of the application and research of this method in animal disease. |
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