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Establishment and application of a fluorescence quantitative Taqman-PCR detection method of mouse poxvirus

Jiangtao DU ; Fangwei DAI ; Shasang ZHOU ; Xiaoming SONG ; Yu LV ; Xiaoying SA

Chinese Journal of Comparative Medicine.2015;(6):59-64. doi:10.3969/j.issn.1671.7856.2015.006.012

Objective To establish a fluorescence quantitative Taqman-PCR method for rapid and accurate detection of mouse poxvirus.Methods After sequence alignment and comparison, ERPV_027 gene was selected as the primer and probe design gene.Furthermore, the specificity, sensitivity, stability and reproducibility of these primers and probes were detected.Results The detection limitation of this method was 68 copies/μL.Data showed that this method has high specificity, which specifically amplifies mouse poxvirus, with no amplification signal of mouse hepatitis virus, Sendai virus, Salmonella and some other viruses and bacteria.This method also showed good stability and reproducibility. Conclusions This study has successfully established a fluorescence quantitative Taqman-PCR method for detection of mouse poxvirus, with high specificity, sensitivity, good stability and reproducibility, and a broad application potential.

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Establishment of a TaqMan real-time fluorescence quantitative PCR for detection of murine polyomavirus

Xueqin YIN ; Wen YUAN ; Jing WANG ; Bihong HUANG ; Dan RAO ; Miaoli WU ; Yujun ZHU ; Shengpeng FENG ; Pengju GUO ; Yu ZHANG ; Ren HUANG

Chinese Journal of Comparative Medicine.2015;(6):53-58. doi:10.3969/j.issn.1671.7856.2015.006.011

Objective To establish a rapid,specific and sensitive TaqMan real-time fluorescence quantitative PCR assay for detection of murine polyomavirus ( MPyV) .Methods The specific primers and TaqMan probe were designed based on genome sequence of MPyV.The primers amplified a 69 bp fragment.After optimizing the reaction system and reaction condition, the standard curve was plotted by detecting recombinant plasmid standards.The specificity, sensitivity and reproducibility of this method were evaluated.In addition, samples of lungs, spleens and feces obtained from experimentally infected mice and 86 clinical samples were used to validate the efficacy of this real-time PCR assay.Results The specificity assay showed that this assay could specifically detect MPyV and the sensitivity for MPyV was about 100 copies/well.The coefficients of variation ( CV) of both intra-assay and inter-assay were less than 1.13%.All of the samples from experimentally infected mice were positive for MPyV and 3 out of 86 clinical samples were positive by this TaqMan-PCR detection with a positive rate of 3.5%.Conclusions The real-time fluorescence quantitative TaqMan-PCR assay established in this study has high specificity, sensitivity and stability.It can be used for clinical diagnosis, routine detection and epidemiological investigation of murine polyomavirus infections.

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Application of suckling mice in pediatric pharmacological and toxicological studies

Chengyu PIAO ; Min YU ; Yongwu LIU ; Shumin LIU

Chinese Journal of Comparative Medicine.2015;(6):71-74. doi:10.3969/j.issn.1671.7856.2015.006.014

Research on laboratory animals is an important issue in biomedicine.Children are a special drug-using population.The selection of suitable experimental animals is a key issue to ensure the scientific quality of research for pediatric drugs.Based on the review of a large number of literature, the authors summarized the application of suckling mice in the pharmacological research and toxicological evaluation of pediatric drugs for the treatment of common diseases in children.We also summarized the existing problems in pediatric toxicology and proposed solutions for providing a reference of test animal application in pediatric drug research.

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Development and application of duplex PCR for detection of H-1 and KRV strains

Xiaobo LI ; Rui FU ; Ji WANG ; Li WEI ; Shujing WANG ; Bingfei YUE ; Zhengming HE

Chinese Journal of Comparative Medicine.2015;(6):46-52. doi:10.3969/j.issn.1671.7856.2015.006.010

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.

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Isolation and chatracterization of microsatellite markers in Tupaia belangeri chinensis

Yuan ZHANG ; Xiaofei LI ; Zhenyu LI ; Pinfen TONG ; Lingxia CHEN ; Bowen YIN ; Jiejie DAI

Chinese Journal of Comparative Medicine.2015;(6):36-41. doi:10.3969/j.issn.1671.7856.2015.006.008

Objective To screen out specific microsatellite markers for use in Tupaia belangeri chinensis genetic testing. Methods Firstly to screen about 700 microsatellite loci from whole genome.Secondly to choose about 100 better loci without defect factors.Lastly 46 primers were designed by 33 tree shrew’ s microsatellite loci obtained from whole genome and other references.Agarose gel electrophoresis and polyacrylamide gel electrophoresis were used for PCR products, and better loci based on electrophoresis results were chosen.Then STR scan was used to select the microsatellite loci combination for genetic testing.Results Twenty-two microsatellite loci were selected with a significant Stutter peak on STR scanning.Comparing the alternative loci and ultimately selected loci, there were two loci available in the five alternative loci of T.glis.The coincidence rate between T.glis and T.b.chinensis was 40%.There were two loci available in the five alternative loci of T.minor, and the coincidence rate between T.minor and T.b.chinensis was 40%.There were two loci available in the three alternative loci of T.belangeri, and the coincidence rate between T.belangeri and T.b. chinensis was about 70%.Conclusions The 22 microsatellite loci screened in this study are well applied for genetic testing of Tupaia belangeri chinensis, therefore, provide a scientific basis for the genetic quality monitoring of tree shrews.

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Susceptibility screening of highly pathogenic porcine reproductive and respiratory syndrome virus in several strains of minipigs

Jinchun PAN ; Bohua REN ; Fangui MIN ; Ruiai CHEN ; Xilong WANG ; Linchuan WANG ; Fengguo WANG ; Shuming LUO ; Jiancong YE ; Ling LIU ; Penghua HUANG

Chinese Journal of Comparative Medicine.2015;(6):14-17. doi:10.3969/j.issn.1671.7856.2015.006.003

Objective To screen strains of minipigs sensitive to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) for evaluation of HP-PRRS live vaccine.Methods Lantang pigs, Juema, Bama and Wuzhishan ( white) minipigs were inoculated with virulent strain NVDC-JXA1 of PRRSV, and local binary hybrid pigs were used as control.The animals were continuously observed for 5 weeks on mental status, appetite, survival, etc.after inoculation of virus.The dead pigs were autopsied and the lung tissue samples were collected for detecting virus by RT-PCR.By the end of the experiment, serum of survival animals were collected for detecting PRRSV antibody by ELISA assay.Result The animals showed depression, anorexia, and other clinical signs and death in each group after inoculation.Meanwhile, the testing results were all positive in the RT-PCR and ELISA detection.Bama and Wuzhishan ( white) minipigs were the most sensitive to virulent strain NVDC-JXA1 of PRRSV regarding mortality rate.Conclusions Bama and Wuzhishan ( white) minipigs are sensitive to HP-PRRSV, and can be used for the inspection of HP-PRRS live vaccine.

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Efficient preparation of a TXNIP knockout mouse model by transcription activator-like effector nucleases (TALEN)

Huanhuan ZHANG ; Chuxin LIU ; Yue MA ; Liping XIAO ; Feida LI ; Huazhong YING ; Huan LIU

Chinese Journal of Comparative Medicine.2015;(6):9-13.

Objective To knockout the murine Txnip gene using microinjection of transcription activator-like effector nuclease ( TALEN) mRNAs.Methods TALEN knockout site recognizing Txnip was designed by tools on line, then constructed the vectors and assayed its cleavage activity at cellular level.TALEN mRNA was transcribed in vitro and microinjected into C57BL/6J mouse zygotes.F0 mice were verified at DNA level with BamHI and TXNIP-knockout mice were obtained.Results We designed and constructed TALENs which recognized and cut the first exon of Txnip, and got four TXNIP knockout mice, among which two were frameshift mutation, demonstrating that the TXNIP-knockout mice were generated by TALEN technique.Conclusions Microinjection of in vitro transcribed TALEN mRNAs into murine zygotes is a highly effective and convenient way to develop TXNIP-knockout mouse model.

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Comparison of Monoamine Neurotransmitters in Two Morphine-dependent Rat Models

Zhixian MO ; Ye LIU ; Jin CHEN

Chinese Journal of Comparative Medicine.2009;19(3):6-9,24.

Objective To investigate the changes of monoamine neurotransmitters of the brain in physical dependence induced by morphine in rats.Methods A physical dependent rat model was established with morphine in a gradually in creasing doses and the withdrawal syndrome was scored after naloxone precipitation.The conditioned place preference(CPP)in rats induced by morphine was used to investigate psychic dependence in rats.Contents of norepinephrine(NE0,dopamine(DA)and serotonin(5-HT)in hypothalamus of rats were assayed with a fluorescent method. Results (1)In naloxone-precipitated withdrawal test of morphine-dependent model rats,after morphine had been withdrawn,morphine-abstinent rats presented marked withdrawal symptoms and signs,their withdrawal scores were significantly increased,and the levels of NE and 5-HT in the rat brain were obviously enhanced,but the content of DA was reduced.(2)In CPP test,morphine caused a marked place preference in rats and the levels of DA and 5-HT in the rat brain were obviously enhanced,but the content of NE was reduced.Conclusiion Morphine dependence development and withdrawal are closely connected with monoamine neurotransmitters in CNS.In the physical dependent model induced by morphine in rats,the rising of NE and 5-HT in the rat brain were significant,but in psychic dependent model induced by morphine in rats,the levels of DA in the rat brain were enhanced p-redominately.

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Therapeutic action of a Uyghur medicine Munziq Balgam on the mouse model of psoriasis

Islam RABIGUL ; Mamat YULTUZ ; Abay SIRAPIL

Chinese Journal of Comparative Medicine.2014;(8):24-27,40. doi:10.3969.j.issn.1671.7856.2014.008.006

Objective To observe the effect of a Uyghur medicine Munziq Balgam on the mouse model of psoriasis.Methods 130 healthy Kunming mice were used in this study .The mouse model of psoriasis was established by applying 5% propranol hydrochloride emulsion to the mouse ear .The tail and ear skin histology was examined using HE staining, and the serum levels of IL-10, IL-17, IL-23 were measured with ELISA.Results The mice of Munziq Balgam treated groups (2, 4, 8 g/kg) did not show obvious abnormalities of general condition and body weight changes (P >0.05).The 8 g/kg Munziq Balgam treated group showed decreased thymus index (P <0.05).The 2, 4, and 8 g/kg Munziq Balgam treated groups showed no significant changes of the serum levels of IL -10, IL-17, and IL-23 (P >0.05). The 2, 4, and 8 g/kg Munziq Balgam treated groups showed promoted formation of epidermis granular layer in the mouse tail skin and improved histology of the ear skin .Conclusions The Uygur medicine Munziq Balgam shows therapeutic effect on experimental mouse models of psoriasis .

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Refining Animal Models to Enhance Animal Welfare

Patricia V.TURNER

Chinese Journal of Comparative Medicine.2012;22(1):1-4. doi:10.3969/j.issn.1671.7856.2012.001.001

The use of animals in research will be necessary for scientific advances in the basic and biomedical sciences for the foreseeable future.As we learn more about the ability of animals to experience pain,suffering,and distress,and particularly for mammals,it becomes the responsibility of scientists,institutions,animal caregivers,and veterinarians to seek ways to improve the lives of research animals and refine their care and use.Refinement is one of the three R's emphasized by Russell and Burch,and refers to modification of procedures to minimise the potential for pain,suffering and distress. It may also refer to procedures used to enhance animal comfort. This paper summarizes considerations for refinements in research animal.

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