Guinea pig as a commonly used laboratory animal is widely used in various fields of biomedical research.The stability of genetic quality directly affects its development and application .Genetic testing is designed to confirm the genetic characteristics of each strain , to verify whether there are genetic mutations and other genetic contamination, to ensure that the test object meets the requirements of this strain .Along with the emerge of biochemical and molecular marker technology , a more convenient and reliable means is provided for research of genetic homozygosity , genetic type detection and genetic quality monitoring of guinea pigs .In this paper, the application and research progress of biochemical, cytological and molecular markers in studies of guinea pig diversity will be summarized , and provide some help for genetic testing guinea pig.

Objective To analyze the population genetics of two outbred colony guinea pigs from two institutions and provide basical information in developing genetic detection methods and standardization of the outbred guinea pigs.Methods 25 polymorphic microsatellite markers were screened by a fluorescent based semi-automated genotyping method for the two populations of guinea pigs, and the population genetic parameters were calculated.Results A total of 121 alleles were detected in the two populations, with 2-10 alleles and a mean 4.84 alleles at each locus.The mean expected heterozygosity was 0.6067, and the average polymorphism information content was 0.552.In the two populations, 103 and 116 alleles were detected, the mean expected heterozygosity was 0.5195 and 0.5838, and mean polymorphism information content was 0.459 and 0.518, respectively.Five loci and six loci, respectively, showed significant deviation from Hardy-Weinberg equilibrium (P<0.05) in the two populations, mostly resulted from heterozygote deficiency.The average Fst of all loci was 0.1056, which implied a moderate genetic differentiation between populations.The Nei’ (1972) genetic distance and Nei ‘(1978) unbiased genetic distance between the two populations were 0.3302 and 0.3204, respectively.Conclusions Both the two populations are consistent with a closed group of animal population genetic characteristics.Several loci deviate from HWE, which probably indicates that a certain degree of inbreeding phenomenon exists during the breeding process.

Objective To analyse and monitor the genetic quality of closed colony NIH mice in Beijing district for the last 3 years.Methods We use biochemical genetic markers( including alkaline phosphatase -1 and the like 14 biochemical markers), selecting A and B colonies from different facilities for genetic monitoring in 2011 to study the polymorphism.And in 2014, 30 NIH mice just from B colony were monitored using the same testing and sampling methods.Results In 2011,NIH mice form both A and B facilities existed 6 polymorphic biochemical markers(Ce2,Car2, Gpi1,Es10,Gpd1,Pgm1); and NIH mice of B company also existed polymorphism in Es3 loucs.Between the 2 NIH mice colonies, there were significant difference in Es3、Gpd1、Pgm1 loci (P <0.01), and difference in Car2 locus(P <0.05).FST of the 2 colonies was 0.0406, the genetic identity was 0.9619, and the genetic distance was 0.0388.In B company, NIH mice of 2014 appeared 2 homozygous loci(Ce2 and Gpd1) when compared with NIH mice of 2011.Between the 2 NIH mice colonies, there were significant difference in Es10 and Gpd1 loci (P <0.01), and difference in Pgm1 locus(P <0.05).Fst of the 2 colonies was 0.1103, the genetic identity was 0.8847, and the Genetic distance was 0.1266.Conclusions Population isolation, breeding and selection, populationpopulation quantityquantity and generation significantly affected the genetic architecture of NIH mice.So when breeding and reserving seeds, we should strengthen the genetic monitoring of outbred NIH mice, in order to offer reliable genetic quality protection.

Objective To develop RT-PCR for detection of TMEV and apply the method .Methods To design specific primers on the basis of GD VII ( GI:62039) genome sequences published in NCBI and establish RT-PCR.To verify the sensitivity and specificity of method after optimizing PCR .We infected 9 BALB/c mice intracerebrally and collected brain, heart, liver, spleen, lung, kidnet, cecal contents and serum samples the 6th day postinfection.The samples were tested by the TMEV RT-PCR.100 mouse cecal contents samples were also detected to apply the established method . Results The 371bp single band was amplified using GDVII as template .Sensitivity test showed that the RT-PCR method can detect as low as 0.69 pg/μL GDVII cDNA.There were no objective band amplified when encephalomyocarditis virus , lymphocytic choriomeningitis virus , Japanese B encephalitis virus , murine norovirus and normal mouse brain tissue were used as case-control .All infected mice showed symptom of different degrees such as depression and hind limb paralysis the 3th day postinoculation and two of infected mice died the 5th day postinoculation.Tissues such as heart, liver, spleen, lung, kidney, brain, cecal contents and serum were collected and tested for TMEV .All the brain samples were detected positive for GDVII and other tissues were all negative;The 100 cecal contents samples were tested and all were negative . Conclusions RT-PCR for TMEV GDVII strain can detect virus infection in mouse tissues efficiently and can be used as a powerful supplement for the national standard of lab animal .

Objective To establish a real-time fluorescent quantitative PCR ( Q-PCR) method for detection of feline herpesvirus 1 ( FHV-1 ) in experiment cats and clinical sick cats.Methods Primers and TaqMan probes were designed and synthesized according to the published FHV-1 specific sequences of TK gene.FHV DNA standards were prepared using molecular biological techniques.The linearity, specificity, sensitivity, stability of the established Q-PCR method were tested.The method was used to detect 48 samples of cats.Results The linear range was 102 copies/μL to 109 copies/μL.The developed Q-PCR method showed no cross reaction with herpes virus type 1 ( HSV-1 ) , canine herpesvirus (CHV), pig pseudo rabies virus (PRV) and cat parvovirus (FPV).The sensitivity was 10 copies/μL.The coefficient of variation ( CV ) was less than 5%.There were 33 positive cases detected in the 48 samples of cats. Conclusions The developed Q-PCR method is good in linearity, specificity, sensitivity, stability, and may be used for rapid quantitative detection of FHV-1 in cats.

Objective To establish and apply an effective PCR assay for detection of the Tupaia ( tree shrew) adenovirus ( TAV) .Methods According to NCBI Genbank, TAV genome DNA from 19418 to 19917 were synthetized and inserted into a plasmid as positive standards.One pair of primers was designed based on this sequence.Sixty blood samples and fifty-six stool samples from tree shrew were detected with this PCR assay.Results A PCR method for detection of TAV was successfully established, with a high specificity and the sensitivity was 13.5 ×10 -7μg/mL.The PCR results of testing sixty tree shrew blood DNA samples were negative.24 positive cases were tested among 56 stool DNA samples.Sequencing of the samples confirmed a 42.9%infection rate of TAV in tree shrew stool samples, well consistent with the PCR results.Conclusions The PCR method for detecting TAV established in this study has good specificity and high sensitivity, therefore, can be used in conventional detection of tree shrew adenovirus.

Objective To understand the characteristics of minipigs infected withJapanese encephalitis virus(JEV).Methods After the brain tissues were treated, the pig brain tissue treatment solution was inoculated with BHK21 cells.Then, virus culture,indirect immunofluorescence assay, neutralization test, electron microscopic observation, and reverse transcription-polymerase chain reaction (RT-PCR) amplification of the new isolate E segment and PrM segment nucleotide sequence were performed and the genotype was identified.Results BHK21 cells were inoculated into 25 pigbrain tissues.Among them, three tissue-treated fluid couldinduce shrinkage and aggregation of BHK21 cells, and immunofluorescence staining showed strong green fluorescence response.The results of neutralization test showed that the neutralization titer of these three new isolates was 1:64, and the size of the virus particles was about 40nm under the electron microscope.The homology of both RT-PCR product sequencing results and E-segment of vaccine strain were 95%.Three new isolates were type GIII JEV.Conclusion The results ofthisstudydemonstrate that there is G III type Japanese encephalitis virus infection in the minipig farm.

Objective Through the detection of rabbit hemorrhagic disease virus( RHDV) antibody, to investigate the capacity of experimental animal quality control laboratories, so as to improve their detection proficiency.Methods According to the program approved by CNAS, the screened samples were numbered randomly and tested for their stability and homogeneity.The random samples were issued to the participant laboratories with the Standard Operation Procedure ( SOP) .The participant laboratories must submit the test reports and original records in time.The feedback results were judged by the rate of concordance with the anticipated results.Results Twenty laboratories from 14 provinces were en-rolled in the evaluation, and all of them submitted detection results on time.ELISA methods were used in 14 laboratories, and hemagglutination inhibition ( HAI) assay was used in 6 laboratories.The results of 17 laboratories were marked as pass or excellent, with a rate of pass of 85%.Conclusions The ability for detection of RHDV antibody in animal test labora-tories in China is high.The implementation of capacity testing can reflect the level of quality control laboratories.

Objective To establish multiplex PCR assay for detection of chicken embryo lethal orphan virus (CELO)and egg drop syndrome virus (EDS).Methods According to GenBank gene sequence , two pairs of specific primers designed were amplified CELO long fiber protein and EDS hexon protein gene sequence .The specificity and sensitivity of multiplex PCR were tested .We also use the multiplex PCR to detect exogenous CELO and EDS in influenza virus.Results Two target bands have been successfully amplified and verified by sequencing .The specificity of the method is better , and the sensitivity is 10 -4μg/mL.The results of detecting exogenous CELO and EDS in 12 influenza virus were negative .Conclusion The multiplex PCR assay for detection of CELO and EDS was established successfully , which have good specificity and high sensitivity , and have high value and application prospect for detecting exogenous CELO and EDS in influenza virus .

Objective To develop a duplex PCR assay for detection of rat parvovirus H-1 and KRV and its application.Methods To design specific primers on the basis of H-1 ( NC_001358 ) and KRV ( U790330 ) genome sequences published in NCBI and establish a duplex PCR assay using H-1 and KRV DNA as templates.To verify the sensitivity and specificity of the method after optimizing PCR.The rats were infected by oral inoculation.The rats were divided into three groups:H-1 infection, KRV infection and mixed infection groups.To collect feces at the 4th, 6th, 8th and 10th days postinfection.Rats were euthanized on the 10th day and samples from heart, liver, spleen, lung, kidney and cecal contents were collected from each rat, then all the samples were screened with the duplex PCR.Results The 183 bp and 302 bp bands were amplified using H-1 and KRV as templates.The sensitivity test showed that the PCR method can detect as low as 3.8 pg/mL H-1 and 0.73 pg/mL KRV.There were no bands amplified when mouse minus virus, canine parvovirus and feline parvovirus were used as templates, showing that the specificity of the duplex PCR assay is very good. The nucleic acids of H-1 or KRV were detected in all rat feces on the 2th day postinfection and there was no obvious clinical symptoms in all the infected rats.The positive rates of H-1 were as follows:50%(4/8) heart tissues, 50%(4/8) liver tissues, 62.5%(5/8) spleen tissues, 50%(4/8) lung tissues, 37.5%(3/8) kidney tissues and 62.5%(5/8) cecum contents, and the positive rate of single infection group was higher than that of mixed infection group.The positive rates of KRV were as follows:0 (0/8) heart tissues, 25% (2/8) liver tissues, 87.5% (7/8) spleen tissues, 12.5% (1/8) lung tissues, 25%(2/8) kidney tissues and 62.5%(5/8) cecum contents, and the positive rate of mixed infection group was higher than that of single infection group.Conclusions The duplex PCR assay for H-1 and KRV established in this study can effectively detect H-1 or KRV infection in rat feces and other tissues, and can be used as an effective supplement to the national standard of lab animals.