Multiple PCR for Detection of Subclinical Mastitis in Dairy Cow

[ Objective] To develop a rapid, sensitive, specific and accurate diagnostic method for subclinical mastitis in dairy cow. [ Method] A total of 70 samples of suspected clinical mastitis cows were detected in gene level by multiple PCR. [Result] The 58 samples were positive with the developed multiple PCR method and the positive rate was 82.5% （58/70）. The 53 samples were positive with the traditional biochemical method and the positive rate was 75.5% （53/70）. As a result, the coincidence rate of both methods was 92%. [ Conclusion] The multiple PCR method is rapid and specific for the detection of pathogenic subclinical mastitis in dairy cow.

Genomic selection of orange-spotted grouper(Epinephelus coioides)based on multiplex PCR enrichment capture sequencing

Orange spotted grouper(Epinephelus coioides)is an important mariculture fish,and genomic breeding of this grouper species has been hindered due to lack of efficient genotyping tools.Here,we developed a single nucleotide polymorphism(SNP)genotyping technology based on multiplex PCR enrichment capture sequencing,which mainly aims at target area for high-throughput sequencing,and 741 SNPs were designed for genomic selection(GS)of growth and ammonia tolerance traits at the same time.The multiplex PCR enrichment capture sequencing assay showed that the genotyping efficiency was more than 99%in the orange-spotted grouper and the predictive accuracy of body weight and ammonia tolerance traits was 82%and 96%,respectively.More importantly,the average identity of the sequences with these SNPs aligned to the genomes of giant grouper(E.lanceolatus)and brown-marbled grouper(E.fuscoguttatus)were both over 96%.Test data showed that the SNP genotyping efficiency was more than 94%in both giant grouper and brown-marbled grouper.In summary,these results indicated that the development of SNP loci and genotyping approach based on the multiple PCR enrichment capture sequencing are suitable for GS of growth and ammonia tolerance traits in various grouper species,and it would provide technical support for practical grouper breeding.

Multiple genotyping based on multiplex PCR and microarray

The genetic variability has obtained more and more attention in the process of diagnosis and treatment of tumors. Herein, we have described a multiple genotyping method based on magnetic enrichment- multiplex PCR （MEM-PCR） and microarray technology. Monodisperse magnetic beads were fabricated and modified with streptavidin. Four loci on two genes （M235T and A-6G loci on AGT gene, A1298C and C677T loci on MTHFR gene） were selected to study single nucleotide polymorphisms （SNP）. Target sequences of these SNP loci were amplified using Cy3-1abeled primers through multiplex PCR in one tube after the templates were enriched and purified by functional magnetic beads （MB）. Four pairs of NH2- labeled probes, corresponding to each locus, were fixed on CHO-modified glass slide by covalent binding. Hybridization between target sequences and probes was performed under suitable conditions. The spotting locations on microarray and the ratio of fluorescence intensity, produced by different loci, were used to distinguish the SNP genotypes. Finally, three of gastric cancer samples were collected and genotvping analysis for these four SNP loci was carried out successfully simultaneously by this method.

PROSPECTIVE STUDY OF MULTIPLE GENETIC TUMOR MARKER ASSAY BY QUANTITATIVE REAL-TIME PCR TO PREDICT RECURRENCE IN COLORECTAL CANCER PATIENTS

Objective To describe correlation between multiple genetic tumor markers,carcinoembryonic antigen (CEA),cytokeratin 20 (CK20),and Survivin,and clinicopathological features of colorectal cancer (CRC) and to assess prognostic diagnosis value in cancer recurrence and metastasis.Methods A total of 92 patients with CRC,68 patients with precancerous lesions,and 29 control volunteers were collected for the detection of CEA,CK20,and Survivin expressions by using quantitative Real-Time PCR technology.Associations among these measurements and clinicopathological features of CRC,and cancer recurrence and metastasis rates in 4-year follow-up were analyzed.Results No mRNA expressions of CEA,CK20,or Survivin were detected in the control group.Expressions of CEA,CK20,and Survivin were 41.3%,47.8%,and 72.8% in CRC patients,respectively.The expressions of genetic tumor markers were related to the clinical stage and lymph node metastasis.In patients with Survivin high expression,4-year survival rate was significantly lower than that in Survivin low expression.The multiple tumor markers assay for CRC patients showed higher specificity and positive detection rate than single marker assay.Patients with CEA,CK20,and Survivin simultaneous expressions had significantly higher 4-year recurrence rate and death rate than those with only one or two markers expression.ConclusionMultiple tumor markers assay including CEA,CK20,and Survivin in peripheral blood by quantitative Real-Time PCR can be an ideal method for the surveillance of the recurrence and prognosis for CRC patients.

青岛地区兔源产气荚膜梭菌的分离鉴定及遗传多样性分析

兔的梭菌性肠炎是由产气荚膜梭菌感染引起的严重危害养兔业的一种疾病,为了更好地控制此病,本研究调查了青岛地区规模化兔场爆发此病时产气荚膜梭菌的毒素型及遗传多样性。2010年11月-2012年5月期间,采集青岛地区规模化养兔场疑似产气荚膜梭菌感染兔的肝脏进行产气荚膜梭菌的分离鉴定,采用Multiple-PCR方法对分离菌株进行毒素型分析,应用ERIC-PCR方法分析分离菌株的遗传多样性。共分离到25株产气荚膜梭菌,其中A型24株(96%),C型1株(4%)。用ERIC-PCR方法将25株分离株分于9个聚类中,其中Ⅴ型为主要流行型。结果表明:青岛地区规模化兔场中产气荚膜梭菌流行的毒素型主要为A型,且具有多种基因亚型,其中Ⅴ型为主要流行型。此结果为该地区兔产气荚膜梭菌病的免疫和微生态防治提供了重要的参考依据。