To monitor and analyze Cricetulus migratorius fungal diversity ,60 adult Cricetulus migratorius brought from Xinjiang region of China were dissected after being euthanized and the specimens were collected .Fungal diversity research was carried out by TaqMan MGB probe real-time fluorescence quantitative PCR ,the ribosome cloning sequencing ,and fungal cul-ture identification techniques .The 60 fungal isolates were characterized from Cricetulus migratorius ,including Candida albi-cans ,Trichosporonasahii ,Aspergillus fumigatus ,Aspergillusniger ,Aspergillussydowii ,Aspergillus japonicus ,Asper-gillus ustus ,Aspergillus versicolor ,Penicillium chrysogenum ,Paecilomyces variotii ,Penicillium aurantiogriseum ,Neuros-pora sitophila ,Neurospora intermedia ,and Cladosporium cladosporioides .Many of them associated with grey hamster as zoonotic pathogens .The results showed that the most dominant fungal group was Aspergillus ,and Penicillium followed by it . These fungi were susceptible to nystatin ,clotrimazole and voriconazole .It’s indicated that application of TaqMan MGB probe real-time fluorescence quantitative PCR ,the ribosome cloning sequencing ,and fungal culture identification techniques could ef-fectively analyze Cricetulus migratorius .The results could provide a scientific basis for Cricetulus migratorius microbiological monitoring and quality standard establishment in China .Overall ,the findings of the present study constitute ,to the authors’ knowledge ,the first extensive report on the diversity of fungal flora associated with Cricetulus migratorius .

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 Giardia lamblia is an important pathogen of zoonosis giardiasis , it poses a potential threat to the quality of SPF (specific pathogen-free) laboratory animals cannot be ignored.The aim of this study is to establish the method of rapid diagnosis of Giardia lamblia, and analyze the test results of 516 batches form 17 manufactures.Methods Direct microscopy, Giemsa-fast staining and multiplex polymerase chain reaction (multiplex PCR) were applied to detect Giardia lamblia.Results Numerous of Giardia lamblia trophozoites and cysts were detected in SPF laboratory animals by using direct microscopy and Giemsa-fast staining, and multiplex PCR were performed to identify 18S rDNA,β-giardin, TPI and GDH genes of DNA extracted from these trophozoites and cysts identified Giardia lamblia.Direct microscopy, Giemsa-fast staining, and multiplex PCR methods can be used to detect Giardia lamblia.Of the 2562 SPF laboratory animals studied, 22.9%(586/2562) were positive for Giardia lamblia.Conclusions Direct microscopy , Giemsa-fast staining , and multiplex PCR were effective techniques with high sensitivity and specificity for rapid diagnosis of Giardia lambliain.It is not satisfactory that the results of Giardia lamblia examination in 516 batches form 17 manufactures failed to meet the requirements 100%.

Objective To develop a TaqMan MGB probe-based,sensitive and specific real-time fluorescence quantitative PCR assay for rapid detection of Helicobacter hepaticus.Methods Primers and probes specific toflaB gene of Helicobacter hepaticus were designed.A TaqMan MGB probe-based,real-time fluorescence quantitative PCR was established.The specificity,sensitivity and stability of the assay were assassed.Then,the established TaqMan MGB probe real-time fluorescence quantitative PCR assay was applied to detect Helicobacter hepaticus in 1081 clinical specimens during 2008-2011,compared with bacterial isolation and culture method and conventional PCR assay.Results The specificity of this established TaqMan MGB probe-based real-time fluorescence quantitative PCR was high and there were no cross-reactivity with Helicobacter pylori,Campylobacter jejuni,Clostridium piliforme,Pasteurella pneumotropica,Escherichia coli,Pseudomonas aeruginosa.The detection limits was 8.3 copies.The correlation coefficient and slope value of standard curve were 0.999 and -3.227,respectively and the efficiency of TaqMan MGB-based probe realtime fluorescence quantitative PCR assay was 100％.The TaqMan MGB-based probe real-time fluorescence quantitative PCR and conventional PCR were preformed to detect Helicobacter hepaticus in 1081 clinical specimens,a total of 86 specimens were positive for Helicobacter hepaticus.However,there was only 4 specimens were positive by bacteria isolation and culture method.The results showed that TaqMan MGB -based probe real-time fluorescence quantitative PCR for Helicobacter hepaticas was more sensitive than bacteria isolation and culture method,and it could detect Helicobacter hepaticus DNA from clinical specimens directly,and detection time is only 2 hours.Conclusion The TaqMan MGB-based probe real-time fluorescence quantitative PCR assay was a reliable,specific,sensitive and useful tool for rapid detection of Helicobacter hepaticus.

Objective To test the potency of hepatitis B vaccine in China. Methods Two inbred strains(DBA/1 and BALB/c) and two NIH closes-colonies of mice were typed in the H-2 region by microcy-totoxicity method and PCR. Groups of mice of the tested strains were immunized with the same hepatitis B vaccine, the titre of anti-HBsAg antibody was analyzed by microplate, and the ED50 was then estimated by Karder method for each strain. Results Significant differences were found between potency estimates de-rived from assays using different strains of mice. Conclusion It is likely that the variation of immune re-sponse to hepatitis B vaccine in mice is correlative with the H-2 haplotype. In some special case, the bet-erozygosity in H-2 region found in NIH stock could influence the accuracy in such testing even a reference preparation of hepatitis B vaccine was used. Base on our experiment, to select an appropriate NIH stocks with the H-2q haplotype for potency testing of hepatitis B vaccine in China.

Objective To investigate the detection capacity of esterase-3 ( Es-3) in the laboratory animals monito-ring laboratories in China, and to improve the quality management of laboratories.Methods We prepared the test sam-ples according to the criteria of China National Accreditation Service for Conformity Assessment(CNAS), all the samples were certificated by homogeneity test and stability test.Then, samples with random numbers and standard operation instruc-tion were distributed to the participant laboratories.The laboratories should submit their reports before the deadline expires. When the results are the same as the standard results, the laboratories will receive excellent remark; when the results are the same as the standard results except the hybridization type, the laboratories will receive satisfactory remark;otherwise, it will receive unsatisfactory remark.If a laboratory did not submit report, the laboratory will also receive unsatisfactory re-mark.Results Ten laboratories participated in the program, and no laboratory received excellent remark.Nine laboratories (90.0%of enrolled laboratories) had satisfactory results, while one laboratory (10.0%of enrolled laboratories) had un-satisfactory results.Conclusions The nationwide overall detection level of laboratories in Es-3 is relatively high.Howev-er, some details should be noticed and several laboratories should improve their detecting ability.

Objective To investigate the detection capacity of malic enzyme 1 and isocitrate dehydrogenase 1 ( Mod1&Idh1) in the laboratory animal monitoring laboratories in China in order to understand the detection capacity of la-boratories and to improve the detection level of laboratory animals’ quality.Methods Based on the program approved by CNAS, samples preparing, homogeneity test and stability test of malic enzyme 1 and isocitrate dehydrogenase 1 in the mouse kidneys were carried out.Standard operation procedure and samples with random numbers were distributed to the la-boratories.The laboratories should submit the result reports before the time limit expires.If the laboratory reports were the same with the standard results, the laboratories will receive satisfactory remark.If laboratory reports were not the same with the standard results, the laboratories will receive unsatisfactory remark.If a laboratory did not submit report, the laboratory will also receive unsatisfactory result.Results Eight laboratories out of 10 (80%) enrolled laboratories reported satisfac-tory experiment results, and two laboratories (20%) presented unsatisfactory results.Conclusions The whole detection level of laboratories in Mod1 &Idh1 is relatively high in the laboratory animals monitoring laboratories in China.It can re-flect the detection level of laboratories to conduct the laboratory capacity evaluation.

Objective To strengthen the quality control management and enhance the detection capacity of the experimental animal quality control laboratoriesin our country through the detection of serum esterase-1 (Es-1) in the experimental mice.Methods The samples were prepared according to the standard procedure, and then were randomly numbered and distributed to participating units by cold-chain transport.Before the deadline, the participants submitted the results and the copies of original records.When the results were completely consistent with the standard results,the results were regarded as satisfactory, otherwise were unsatisfactory.Results A total of 11 laboratories participated in this program, of which 10 laboratories were regarded as satisfactory (90.9%) and one laboratory obtained unsatisfactory result (9.1%).Conclusions The results of this proficiency testing project demonstrate that the overall detection level of Es-1 in laboratory mice is highof the participating laboratories.However, more attention still should be paid to standard specifications and some test details.

Objective To develop a multiplex polymerase chain reaction ( mPCR) assay for detection of four pathogenic dermatophytes [Trichophyton mentagrophytes (Tm), Microsporum gypseum (Mg), Microsporum canis (Mc), and Arthroderma simii ( As) ] in laboratory animals, which could be used rapidly and simultaneously for direct detection of those four pathogens.Methods We designed 5 specific primers according to 18S-28S rRNA sequences of the four pathogenic dermatophytes reported in Genbank. The four mPCR assays were established through optimizing the concentration of primers, dNTP, TaqDNA polymerase and the annealing temperature.After verifying the specificity and sensibility, this method was used to detect 15 hair samples with artificial infection and 260 samples taken from laboratory animals.Results This mPCR technique can distinguish the four dermatophytes by producing 192 bp( Tm) ,460 bp( Mg) , 290 bp( Mc) and 602 bp( As) fragments.The sensibility for detection of the four dermatophytes was 5.9 pg/μL, 6.6 pg/μL, 9.5 pg/μL and 5.1 pg/μL, respectively.The results of 15 artificial infection samples were accurate, and the results of 260 hairs samples were negative for the four fungi.Conclusions Our results suggest that the mPCR assay developed in this study can efficiently detect the four dermatophytes, is a useful and rapid technique for rapid detection of the pathogenic dermatophytes in laboratory animals.

Objective To improve the accuracy of detection through analyzing the phenotypes of P.pneumotropica isolates in laboratory animals in Beijing area .Methods 306 suspicious P.pneumotropica strains were identified by biochemical identification and 16S rDNA sequencing.Then, to obtain the phylogenetic relationships combined with colony characteristics on blood agar plates and biochemical characteristics of 53 biotypes .Results BD Phoenix 100 automated bacterial identification system and 16S rDNA sequencing identified P.pneumotropica positive rate of 306 isolates were 164/306 and 227/306, respectively.There were 140 phenotypes in 227 true-positive strains, of which 106 were biotype Heyl and 23 were biotype Jawetz .Conclusions In the samples of laboratory animals in Beijing area , P.pneumotropica infection mainly are of biotype Heyl , and less is of biotype Jawetz .The phenotypes are diverse and widely distributed .