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 .

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 establish an effective PCR assay for leptospirosis detection , and applicate the assay in tree shrew, mongolian gerbil and gray hamster .Methods Sequence of leptospira was obtained from the NCBI Genbank , and primers were designed based on the sequences .The positive amplified fragments were sequenced to verify the reliability of the method.The samples from tree shrew, mongolian gerbils and hamsters were tested using this PCR method .Results The PCR method for detection of leptospirosis was successfully established .The positive rate of Leptospira was 8.33% in 60 samples of conventional tree shrews , 100% in 104 samples of the conventional Mongolian gerbils , and 0% in 60 samples of clean gray hamsters.Conclusions The establishment of this PCR assay is useful in the detection of leptospirosis in tree shrew, mongolian gerbil and gray hamster .The results of our investigation of leptospira infection levels of the three new experimental animals may promote their application in biomedical research .

Objective:we aim to determine the relationship between Cell sarcoma (c-Src) expression in patients with EOC and the disease phenotype.Methods:c-Src expression was evaluated using Western blotting analysis in 21 ovarian carcinomas and 4 normal ovarian tissues.Immunohistochemistry was used to evaluate c-Src expression in 134 ovarian carcinomas and 26 normal ovarian tissues.The association between c-Src expression and clinically pathologic characteristics were also assessed in these patients.Results:Our results indicated elevated c-Src protein in EOCs compared with that in normal tissues.The overexpression of c-Src was significantly associated with aggressive features,such as advanced disease stage,poor histological grade,lymph node metastasis,and tumor recurrence (P＜0.05).In addition,the overexpression ofc-Src is significantly associated with EOCs' prognosis.Conclusion:c-Src overexpression was significantly associated with the malignant biological behavior of tumor,suggesting c-Src as a potential preventive target in these patients.

Objective To explore the effect of electromyogram-triggered neuromusclar stimulation (ETNS) on motor function of upper limbs of stroke patients. Methods 45 stroke patients from July, 2011 to December, 2012 in China Rehabilitation Research Center were randomly divided into control group (n=15), neuromuscular electrical stimulation (NMES) group (n=15) and ETNS group (n=15). 3 groups were given routine medication and rehabilitation treatment. They were assessed with the largest surface electromyography (sEMG), Simple Test for Evaluating Hand Function (STEF), and modified Barthel Index before and after treatment. Results After treatment, the range of sEMG of extension carpi radialis and STEF improved in three groups (P<0.05). NMES group and ETNS group were better than the control group (P<0.05), and ETNS group was better than NMES group (P<0.05). The scores of modified Barthel Index rose (P<0.05), NMES group and ETNS group were better than the control group (P<0.05). Conclusion Both NMES and ETNS can improve the motor recovery of upper limbs after stroke, and ETNS is more effective.

Background The integration of segregated pathways from the two eyes first appears in V1 neurons,where it not only plays a critical role in the generation of a three-dimensional visual representation.Abnormal visual experiences in critical period usually lead to amblyopia and binocular integration defects.Objective Present study was to investigate how neurons of kitten coordinate their activity patterns in response to synchronous dichoptic stimulus inputs in striate cortex.Methods Spike rate and local field potential(LFP) gamma band(20-90Hz) power of three kitten(1-1.2Kg,8-10 weeks old) to monocular and synchronous dichoptic presented gratings were assessed for 28 binocular neurons in V1 of kitten by in vivo extracellular record method under anaesthesia and paralysis.Ocular dominance index(ODI) and binocular integration index(BII) were assessed and the correlation between these two indexes were analyzed.Results In 28 cells with binocular characteristic,the absolute value of spike-ODI was significant larger than that of LFP-ODI(t=2.606,P=0.021).A positive linear correlation between the ocular preferences of spike and LFP was found(R2=0.513,F=27.423,P=0.003).In dichotic trails,binocular facilitation with BII for spike was 2.348±0.996,showing a significant reduce in comparison with BII for LFP(3.678±1.974)(t=2.671,P=0.019).Binocular integration index for two signals were greater when monocular responses of both eyes were similar(P=0.035 and P=0.124,respectively).Conclusion Both spike rate and gamma band power of LFP exhibited binocular facilitation to synchronous presented dichotic stimuli with significant facilitation induced by balanced monocular responses.Spiking activity and LFP reflect neural activities of different spatial scales and source components.

Objective To understand the Salmonella detectability in the laboratory animal testing laboratories, im-prove the level of detection for the quality of laboratory animals, by means of laboratory animals Salmonella proficiency tes-ting program.Method According to the proficiency testing program approved by CNAS, freeze-dried animal stool samples containing Salmonella bacteria and interference bacteria were prepared, and through stability and homogeneity tests quali-fied as proficiency testing samples.The randomly numbered samples were issued to the participating units by cold-chain transportation, and attached work instructions.The original reports and copies of the tests should be submitted on time.The sample results consistent with the results of the pretested results were considered as satisfactory, and the results inconsistent or fails to submit were judged as unsatisfactory.Results A total of 30 laboratories from 20 provinces and cities nationwide participated in this proficiency testing programs for Salmonella, including 28 ( 93.3%) laboratories with satisfactory re-sults, and two laboratories unsatisfactory ( 6.7%) .29 laboratories used separate culture methods, and two laboratories used PCR method.Conclusions The laboratory animal quality inspection agencies have good detection ability for Salmo-nella.The implementation of the capacity verification plan can well reflect the detection level of laboratories.

Objective To verify the detection ability of experimental animal quality detection laboratories in China for Staphylococcus aureus.Methods The testing samples for Staphylococcus aureus detection were prepared by bacterial culture, homogeneity test and stability test, according to the study plan approved by CNAS.Then the samples and operation instruction were sent to the participant laboratories.The detection reports from these laboratories should be submitted before the deadline expires, and the collected data were summarized and analyzed.Results There were 28 laboratories which joined to this test plan.Among them 22 laboratories ( 78.57%) achieved satisfactory test results, and six laboratories (21.43%) had unsatisfactory test results.27 Laboratories used the national standard detection assay, while only one labo-ratory used PCR assay.Conclusions Most of experimental animal quality testing laboratories in China have sufficient pro-ficiency in detection of Staphylococcus aureus.The obtained information are very helpful for the laboratory ability verification testing in future.

Objective We established a rapid detection method of Pasteurella spp.and provided a reference for microbiological quality control of laboratory animal .Methods According to the β subunit of bacterial RNA polymerase ( rpoB) protein multiple alignments of 13 different Pasteurella spp.published in NCBI .The degenerate primers were designed by CODEHOP designer online .CODEHOP PCR method was applied to detecting Pasteurella spp.after the specificity and sensitivity of the method had been evaluated by 21 reference strains .Results Standard strain amplified fragment were about 200 bp by degenerate primers PastF6/PastR5.The primers are able to distinguish between Pasteurella spp.and the other pathognic organisms of laboratory animal respiratory tracts .Sensitivity of this method were 0.2 pg/μL~2 pg/μL to different Pasteurella.The Pasteurella positive rate was 19.1% in 609 animal ' s respiratory samples .The accuracy of positive results was 100%through verifying by sequenced and blast .Conclusions The established method has good specificity and sensitivity .It can be used to detect Pasteurella spp.in animal samples .