ABSTRACT
Objective To study the relationships between differences in tumorigenicity and immu-nogenetic backgrounds of nude mice. Methods According to the Chinese Pharmacopoeia, positive and neg-ative groups were set up in both Laboratory A and B with ten nude mice in each group. Organ tissues were collected for clinicopathological analysis. Blood samples were collected and detected using flow cytometry. DNA was extracted and analyzed with 23 STR markers. Results The positive group of Laboratory B was in-valid (7/10 tumor formation). The two laboratories showed no significant difference in the results of patho-logical analysis, but had significant differences in CD25, CD8, CD4, Th1 and Th2. There were 13 and 18 polymorphic sites respectively found in nude mice of Laboratory A and B. Further analysis of the non-tumor-bearing nude mice in Laboratory B positive group revealed that CD25, Th2, D3Mit29 and D5Mit48 were the specific indexes. Conclusion Differences in tumorigenicity might be related to the diversity of immunoge-netic backgrounds of nude mice.
ABSTRACT
Objective To establish and preliminarily apply an effective PCR assay for detection of Tupaia(tree shrew)paramyxovirus(TPMV). Methods Using TPMV genomic DNA from NCBI GenBank, bases 8231 to 8720 were synthesized and inserted into a plasmid as a positive standard. One primer pair was designed based on this sequence. In total,60 respiratory swabs and 12 lung tissues from the tree shrews were tested in this PCR assay. Results A PCR method for detection of TPMV was successfully established,with high specificity and sensitivity of 11.5 × 10 -5μg/mL. PCR result testing 60 respiratory swabs and 12 lung tissues were negative. Conclusions PCR for detecting TPMV has good specificity and high sensitivity and can be used for conventional tree shrew paramyxovirus detection.
ABSTRACT
Objective To analyze the result of proficiency testing(PT)of detection activities for Laboratory animal pathogenic bacteria in 2011 and 2013-2017. To further improve the detection capacity of laboratory animal testing agency,and promote PT to be carried out in future. Methods During the six years(2011 and 2013 -2017), the National Institutes for Food and Drug Control conducted a total of six(seven projects)PT activities of laboratory animal pathogen bacteria. We analyzed the overall trend and the exposed problems by summarizing the result data of the PT in 6 years. Results A total of 45 laboratories in the country including 20 provinces and cities participated in the PT. The PT projects included Mycoplasma pulmonis, Clostridium piliformis, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella spp.,Klebsiella pneumoniae and Bordetella bronchiseptica. The satisfaction rates were 75%,87.5%,80.0%, 78.6%,93.3,96.2% and 88.0%, respectively. The main reasons of unsatisfactory results were for lack of incubation time,select errors of suspicious bacteria, biochemical identification errors, report writing errors and not timely feedback results. Conclusions The level of domestic laboratory animal pathogenic bacteria detection is gradually increased to achieve the desired goal through continuous proficiency testing activities.
ABSTRACT
Objective To establish a rapid, simple, sensitive, and specific multiplex real-time PCR method for quantitative detection of Campylobacter jejuni, Salmonella and Shigella in tree shrews.Methods Specific primers and probes were designed, according to the HipO gene of Campylobacter jejuni, inV gene of Salmonella and ipaH gene of Shigella.The primers were confirmed by single pathogen quantitative PCR, and the sensitivity and specificity of the multiplex PCR were analyzed.Finall, the samples of experimental tree shrews were detected by this multiplex PCR method.Results The PCR element of TaqMan-MGB real-time PCR assay was able to quantitatively amplify the Campylobacter jejuni, Salmonella or Shigella.Appropriate standard amplification curves of Campylobacter jejuni, Salmonella and Shigella in the multiplex quantitative PCR were obtained.The sensitivity of this method was 1×103 ng/μL.There was no false positive detection from other bacterial strains.Conclusions This multiplex quantitative real-time PCR method has good application and development prospects in the detection of microorganisms in tree shrews.
ABSTRACT
Objective To screen the markers of chromosome 12, which is vacant in the Beijing local standard DB11/T828.3-2011 for genetic quality control of miniature pigs in Beijing, and to study the applicability of the local standard by comparison of two different methods for evaluation of the genetic quality of the same population.Methods According to the literature, we selected four pairs of microsatellite markers of chromosome 12 and studied the polymorphism through monitoring the genetic quality of two populations of China Agricultural University miniature pigs.We screened the highly polymorphic markers of chromosome 12, combined them with the microsatellite primers of the standard 18 pairs of chromosomes to establish the whole chromosome method.We compared and analyzed the applicability of the local standard DB11/T828.3-2011 through monitoring the genetic quality of the same population of miniature pigs with different method.The data were processed and analyzed using software Popgen32.Results All the screened four pairs of microsatellite markers of chromosome 12 were highly polymorphic (PIC>0.5).The local standard showed a chromosome coverage of 94.7%, stability of amplification of 96.0%, and certified that the China Agricultural University miniature pig III was qualified, while the China Agricultural University miniature pig I was not qualified.When the markers of chromosome 12 were added, the whole chromosome method showed result of 100% chromosome coverage and 96.6% amplification stability, both of the two populations of pigs were certified as qualified.Conclusions The four screened markers of chromosome 12 are all highly polymorphic, and provide a support for supplement of the local standard DB11/T828.3-2011.
ABSTRACT
Objective To compare and analyze the genetic structure of NIH mice bred in Unites A and B, using microsatellite technology.Methods Thirty SPF 8-week old outbred NIH mice (half male and half female) of each population were randomly chosen from the Units A and B, respectively.PCR amplification and STR scan were performed to determine the genetic characteristics of two outbred populations using microsatellite loci, and the population genetic structure was analyzed with statistical software Popgene 1.32.Results In the NIH mouse population form the Unit A, 74 alleles were obtained, with an average heterozygosity of 0.3108 and polymorphism information content of 0.2637.In the NIH mouse population from the Unit B, 76 alleles were obtained, with an average heterozygosity of 0.3257 and polymorphism information content of 0.2777.The inter-population comparison showed that genetic differentiation coefficient Fst was 0.3932, the genetic identity was 0.3971, and the genetic distance was 0.9235.The population difference was significant.Conclusions There is serious genetic differentiation between the two NIH mice populations,resulting in the formation of two different closed populations.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Objective To establish a detection technique for H.pylori(HP) infection in Mongolian gerbils using nested PCR technique.Methods H.pylori was cultured in vitro and inoculated into Mongolian gerbils.At the 10th week after infection, the HP in the gastric juice of Mongolian gerbil was detected by conventional PCR assay and the gastric juice, gastric mucosa, duodenal contents and colon stool were examined by nested PCR.Rapid urease test and ELISA were used to analyze the accuracy of the nested PCR assay.All of the PCR products were verified by sequencing.Results The positive rate of gastric juice detected by conventional PCR was 30%, while the positive rates of gastric juice, gastric mucosa, duodenal contents and colon stool detected by nested PCR were 100%, 100%, 90%, and 10%, respectively.The positive detection rates of rapid urease test and serum ELISA were 100% and 0%, respectively.Comparing the results of different methods, both the positive rates of gastric juice and gastric mucosa detected by nested PCR and the detection rate of rapid urease test were 100%, but the results of conventional PCR detection of gastric juice, the nested PCR detection result of stool in colon and of serum ELISA assay were lower than other methods.Conclusions Due to its high accuracy and sensitivity, the nested PCR assay of gastric juice can be used for the long-time detection of H.pylori infection in Mongolian gerbils, especially useful in the experiments of prevention and treatment of H.pylori infection.
ABSTRACT
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 .
ABSTRACT
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.
ABSTRACT
Objective To analyze and evaluate the population genetic quality of 3 subbreeds of China Agricultural University miniature pigs in Beijing.Method According to the local standard DB11/T828.3 -2011, 25 pairs of microsatellite primers were used in 3 subbreeds of China Agricultural University miniature pigs, and software Popgen32 was used to process the data.Results 24 microsatellite loci shared 130, 122 and 138 alleles in the China Agricultural University miniature pigs I, II, III, respectively. The average heterozygosity was 0.6759, 0.5967 and 0.6779, respectively, while the average polymorphism information content ( PIC) was 0.6344, 0.5540 and 0.6403, respectively. The genetic distance between China Agricultural University miniature pig II and III was 0.4251, while the genetic distance between China Agricultural University miniature pig I and II was 0.2084.Conclusions In the 3 subbreeds, China Agricultural University miniature pigs II and III have genetic stability and genetic diversity, and both of which satisfy with the genetic characteristics of closed colony laboratory animal.
ABSTRACT
Objective To test and analyze the genetic background of highly immunodeficient mice from different sources.Methods Four highly immunodeficient mouse strains from different sources of NOD background were collected. 30 microsatellite DNA sites were detected, and the genotype can be displayed by gel electrophoresis and STR scanning. Results 17 microsatellite sites exhibit polymorphism in 20 mice of the four groups.There were 30 homozygous loci in the mice of groups A and B, and heterozygous in the other two groups.The genetic distance is minimum between groups A and B, showing a higher genetic similarity.Conclusions The genetic backgrounds are different in highly immunodeficient mice from different sources.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Objective Through the proficiency validation of testing of mammalian orthoreovirus 3 (Reo3)antibody in laboratory mice, to investigate the capacity of experimental animal quality control laboratories, so as to improve the de-tection capacity.Methods According to the program approved by CNAS, serum samples after calibration were tested for stability and homogeneity, and numbered randomly.The random samples were issued to the participant laboratories with the Standard Operation Procedure( SOP) .The participants must submit the test reports and original records in time.The feed-back results were judged by the concordance rate with the anticipated results.Results 27 laboratories from 17 provinces were enrolled in this evaluation project, all of them submitted detection results on time.Results All the 27 laboratories were marked as pass or excellent, with a pass rate of 100%.ELISA was used in 26 laboratories, and immunofluorescence assay was used in one laboratory.Conclusions The ability for detection of Reo3 antibody in laboratory mice in animal test laboratories is high.The implementation of proficiency testing can reflect the inspection level of quality control laborato-ries.
ABSTRACT
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.
ABSTRACT
Objective To acquire the prevalence and molecular identification data on Syphacia muris and provide reference for the revision of national standard. Methods 923 batches of 5199 SPF animals ( including one batch of 5 monkeys, 3 batches of 25 mini?pigs, 28 batches of 55 rabbits, 13 batches of 248 hamsters, 37 batches of 198 guinea pigs, 93 batches of 459 rats, 742 batches of 4179 mice, 5 batches of 25 chickens and one batch of 5 ducks) and 145 batches of 1389 clean animals ( including one batch of 3 rabbits, 4 batches of 31 hamsters, 16 batches of 157 guinea pigs, 32 batches of 268 rats and 92 batches of 930 mice ) came from 50 different manufactures in China. Direct microscopy real?time dynamic video recording techniques in combination with morphological identification method were applied to screen the Syphacia muris infestation. A multiple polymerase chain reaction ( multiple?PCR ) testing of the isolate based on amplification of the conserved portions of the Syphacia muris internal transcribed spacer (ITS), 28S ribosomal RNA (28S rRNA), NADH dehydrogenase subunits 1 (nad1) and cytochrome c oxidase subunit 1 (cox1) genes, and the molecular sequencing of the multiple?PCR amplicons was used to confirm the Syphacia muris infection. Results Syphacia muris eggs, larvae and adults were detected by using direct microscopy real?time dynamic video recording technique. Syphacia muris were detected based on the morphology and size of ovum, larvae, and female and male adult worms. Multiple?PCR and sequencing were performed to identify ITS, 28S rRNA, nad1 and cox1 genes of DNA extracted from the single egg, larva and adult parasite Syphacia muris. This approach allowed the specific identification with no amplicon being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the amplified sequences. Molecular characterization by multiple?PCR amplification and sequencing of the ITS, 28S rRNA, nad1 and cox1 genes demonstrated the presence of Syphacia muris. Multiple?PCR followed by sequencing confirmed 285 of 5199 SPF and 135 of 1389 clean animal samples classified as positive by using direct microscopy real?time dynamic video recording technique in the study as containing Syphacia muris?specific DNA. Comparison of the partial sequences of the ITS, 28S rRNA, nad1 and cox1 genes revealed 100% similarity amongst Syphacia muris from different animals. The prevalence of Syphacia muris infection in SPF and clean animals were 5?5% (285/5199) and 9?7% (135/1389), respectively. Conclusions Direct microscopy real?time dynamic video recording technique, multiple?PCR and sequencing can be used to rapidly detect and accurately identify Syphacia muris. The zoonotic nature of Syphacia muris can be regard as a public health alter, hence the good quality control of animal has an important role in protecting human health and safeguarding people safety. This is the first molecular identification and infection investigation of Syphacia muris in SPF and clean animals in China.
ABSTRACT
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 .