Small RNAs regulate gene expression at multi-level, such as chromosome construction, transcription, translation, RNA modification and stability. piRNA (Piwi-interacting RNA) mainly participates in the process of development of germ cells, scilence of transposon, formation of heterochromatin, DNA integrity of germ cells, and so on. It is significant to steady heredity of hereditary substance. As a novel small RNA, piRNA has received wide attention. Since piRNA was found in 2006, considerable inference, quest and verification about the biogenesis and function of piRNA have been carried out by researchers. Studies found that piRNA mainly distributed in the animal testes spermatogonial cells and ovarian oocytes, and there are also a small number of piRNAs in Drosophila ovarian somatic cells (follicle cells). piRNA is generated by different ways in germ cells and somatic cells. The way in somatic cells is relatively simpler than that in germ cells, the way in germ cells is mainly through the “ping-pong cycle”. In the “ping-pong cycle”, Piwi locate in the nucleus, Ago3 and Aub locate in the cytoplasm. This phenomenon imply the piRNA may generate and play function in different compartments. The 3′ end hypermethylation of piRNA is widespread, which is significant to the stability of piRNA, but not the necessary conditions to determine the length of of mature piRNAs. Known fuctions of piRNA-related proteins are important clues to predict the biogenesis and fuctions of piRNA. At present, some proteins related to piRNA biogenesis and function have been discovered. piRNA expression is limited to germline cells and its regulatory network is more complex than that of siRNA (small interference RNA) and miRNA (microRNA). The emergence of novel methodology and system speed up the research process of piRNA biogenesis and function. This article is a brief overview focusing on piRNA biogenesis, produce and function related proteins and research methods.

Metagenomics, an emerging microorganism-researching technology in recent years, directly focuses on the whole genetic materials of microorganism in all kinds of environment and breakthroughs limitations of traditional research methods, such as isolating culture, serologic reaction and PCR. Viral metagenomics has been used to discover many novel viruses in fields of human guts, animal tissues, blood, and sewer and so on, and help people to understand the virome constitutions in these environments as well as the enlargement of host ranges of many viruses, embodying the pragmatic significance of novel virus discovery, pathogen tracing and bioEW. This review briefly introduced the history of viral metagenomics and its definition and summarized some major applications of this technique and suggested that the analogous research should be carried out more in China.

In order to understand the structure and function of porcine LPL gene, the mRNA expression profile of porcine LPL in five tissues (heart, liver, kidney, subcutaneous fat and longissimus muscle) were examined in Suzhong pigs by Real-time PCR. Besides, the coding sequences (CDS) of LPL were cloned and sequenced in Suzhong pigs, and molecular structure and function of LPL were analyzed by bioinformatics methods. The results showed that mRNA expression abundance of LPL in different tissues tended to be subcutaneous fat > heart>kidney>longissimus muscle>liver. Among these tissues, LPL expression in subcutaneous fat was significantly higher than that in any other tissues (P<0.01), and expression in longissimus muscle of boars was significantly higher than that of sows (P<0.01). There was significant difference of eye-muscle area and lean-meat percentage traits between boars and sows(P<0.05). Bioinformatic analysis revealed that porcine LPL gene encode a protein of 479 amino acids, and its theoretical isoelectric point was 4.99. LPL protein was a hydrophobic and secreted protein with a signal peptide. In addition, Suzhong pig was closed to cattle, sheep and cat in phylogenetic tree. These findings suggest that the LPL may play an important role in regulating fat deposition in tissues of Suzhong pigs, such as subcutaneous fat, heart, kidney and muscle, etc, and it can be an important candidate gene for improving meat quality in Suzhong pigs.

 The study focused on the effect of CART on FSH-induced estradiol production of porcine ovarian follicular granulose cells.Granulose cells were cultured for 7 days under different CART and FSH concentrations by Long-term culture. The growth situation in each well was observed and pictures were taken. The content of estradiol in each well was determined by sandwich ABC-ELISA. After 168 h of incubation, (1) In CART-free medium, the estrodiol levels in wells supplemented with FSH significantly increased than that of FSH-free well. Adding of 25 ng·mL^-1 of FSH resulted in the highest estrodiol level of (17 295.57±184.03) pg·mL^-1; (2) CART showed no effect on estradiol production by porcine ovarian follicular granulose cells cultured in FSHfree and 5 ng·mL^-1 FSH medium; (3) In the medium containing 5 or 25 ng·mL^-1 FSH,with the increase of CART concentration（0.01, 0.1, 1 μmol·L^-1）, the levels of estradiol concentration showed a tendency to decrease but there was no significant differences among the three groups(P>0.05). However, when FSH at 25 ng·mL^-1, CART at 0.1 or 1 μmol·L^-1, compared with the control group (FSH 25 ng·mL^-1, CART 0 μmol·L^-1), estradiol secreted by follicular granulose cells was suppressed markedly(P<0.05). The result of statistical analysis demonstrated no significant interactive effect between FSH and CART (P>0.05), which needs further investigation. (4) When the medium contained 50 ng·mL^-1 of FSH, the increase of CART concentration showed no significant influence on estradiol concentration in different groups (P>0.05). The production of follicular estradiol in pig was induced by FSH ; when the concentration of FSH were 25 and 50 ng·mL^-1, CART inhibited the estradiol production by porcine follicular granulosa cells, but no significant inhibitory effects.The result indicate that CART maybe not a main local negative regulatory factor in pig follicular development.

The aim of this study was to use genome-wide association analysis to explore the candidate genes and molecular markers associated with various economic traits. The GWAS of the body weight at d1,d28,d56,d80,d100 of Beijing-You chicken using the Illumina 60K SNP Beadchip was performed. The results showed that there were 9 SNPs reaching 5% Bonferroni genome-wide significance, which were significantly associated with body weight at d28 and d100, including LYRM1, LDB2 and other genes near these SNPs; There were 96 SNPs reaching the significance of “suggestive linkage”, which were potentially associated with each indexes detected. The result revealed that these genes and SNPs laid a foundation for the development of techniques of marker assisted selection.

 In order to study the roles of miR-184 and miR-205 in hair follicle development of sheep, the expression of miR-184 and miR-205 at three different developmental stages of hair follicle of sheep was detected by QPCR. The precursors of the two microRNAs was predicted and the sequences were mapped to genome of sheep. Furthermore target genes and pathways of the two microRNAs were predicted. The results indicated that the expression of miR-184 in hair follicle tissue showed increasing trend from anagen to telogen. However, the miR-205 was upregulated from anagen to catagen, and then downregulated from catagen to telogen. The expression of miR-205 was highest in catagen. miR-184 and miR-205 both express in many different tiusses. Moreover, the candidate target genes of the two microRNAs are involved in 15 different pathways and miR-184 and miR-205 probably regulate the hair follicle development through Wnt signaling pathway.

 This study aimed to reveal the variations of daily milk yield, milk fat percentage, milk protein percentage and somatic cell score (SCS), and to establish the prediction models for these parameters in the lactation period for Chinese Holstein in southern China. A 33194-test-day dairy herd complete data from 5 Chinese Holstein dairy farms were collected in the southern China from first lactation to third lactation between 2008 to 2010 and fitted to nonlinear curve of test-day milk yield, milk fat percentage, milk protein percentage and SCS with the Wood’s incomplete gamma function model. The curve of test-day milk yield for Chinese Holstein was the standard lactation curve, and the curves of milk fat percentage, milk protein percentage and SCS were the reversed standard lactation curve. The best fitness of the Wood’s model occurred for milk protein percentage and daily milk yield with the lowest residual mean square, then following for milk fat percentage. The poor model fitness (R2≤0.7) was observed for SCS which residual mean square was highest. Daily milk yield peak day was accompanied with occurrences of minimal milk protein and SCS in the estimated lactation model. The minimal milk fat percentage came at the latest time of 18^th week to 21^th week in lactation curve. The peak milk yield was 30.4 kg·d^-1 for first-parity cows, but the persistence for maintaining high milk yield and low SCS were greater than those of second- and third- parity cows in the latter lactation period, and the maximal milk yields for second- and third-parity dairy cows were 35.9 and 36.2 kg·d^-1, respectively. The persistence for keeping high milk fat percentage and milk protein percentage was greater for second-parity cows than those of first- and third-parity cows in the latter lactation period. The Wood’s incomplete gamma function model was appropriate to predict the variation for test-day milk yield, milk fat percentage, milk protein percentage, and was not appropriate for SCS for Chinese Holstein dairy cows in southern China.

Two different microbial markers, ¹⁵N and purine bases(PB), were used in this experiment with the purpose to establish the relationship between the two methods and provide a precise way for measuring microbial N yield in mutton sheep. Twelve Dorper ( Small-tailed Han crossbreds, noncastrated male lambs ((41.3±2.8) kg BW) were randomly assigned to three levels of dry matter intake: ad libitum intake, 70% or 40% of the ad libitum intake, with four lambs at each level. The experiment lasted for 25 d (10 d for adaptation and 15 d for trial period). The ruminal bacterial components (N, purine bases and purine bases/N) were not affected by dietary treatment (P>0.05). Duodenal flows of dry matter, organic matter, non-ammonia nitrogen and PB decreased significantly with decrease of feed intake (P<0.05). Microbial N yield measured with either ¹⁵N or PB decreased significantly with decrease of feed intake (P<0.05) whereas the efficiency of microbial synthesis was not affected by dietary treatment for both markers (P>0.05). The result indicate that microbial N measured with ¹⁵N showed a smaller within-treatment variation than that measured with PB, and thus ¹⁵N can be a reliable marker for accurately quantifying microbial N.

In order to study the pathological changes and the etiological agent of the Swine High Fever Syndrome (SHFS) in central region of Shandong province, we diagnosed the clinical symptoms and the pathological changes of 109 cases in 56 pig farms and collected 363 serum samples from Jan. 2011 to Mar. 2012. Three viruses were detected on tissue samples by histopathological, immunohistochemical examination and RT-PCR or PCR, including classical swine fever virus (CSFV)，porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV-2). Serological tests were used for detecting prevalence of CSFV，PRRS, and PCV- 2 antibody by ELISA. The results showed that all pigs suffering from high fever had been vaccinated by Classical Swine Fever vaccines, part by Porcine Reproductive and Respiratory Syndrome attenuated live vaccines, but none by Circovirus vaccines. However, they had high positive rates of antibody against CSFV，PRRSV, and PCV-2. The pathological changes mainly included acute inflammation in lymphoid tissues, interstitial pneumonia and viral encephalitis, and the occurrences were respectively 92.3%, 76.1% and 66.1%.The pathogenetic rates of CSFV, PRRSV and PCV2 were respectively 30.27%, 66.97% and 41.28%. Co-infection rates of CSFV and PRRSV, CSFV and PCV2, PRRSV and PCV2 were respectively 16.51%, 6.42% and 28.44%. And the triple infection rate was 4.59%. In addition, other pathogenic infections were 8.26%. Sequence analysis showed that the epidemic strains of CSFV were developing towards being far away from the HCLV, PRRSV strains were highly pathogenic ones, and PCV2 strains were mainly the virulent Type-PCV2b. In conclusion, CSFV, PRRSV and PCV2 were the main pathogens which caused the Swine High Fever in central region of Shandong province, and the severe cases were mainly caused by co-infection. What’s more, mixed infection of Mycoplasma and Haemophilus parasuis used to occur in clinical cases. Therefore, the Swine High Fever in the region was due to multiple infection.

This study was designed to compare the indirect ELISA methods for detection of dog antibodies against rabies virus based on the recombinant matrix protein (M) and phosphoprotein (P). The M gene of rabies virus LEP-Flury strain was amplified by PCR and cloned into prokaryotic expression vector pGEX-6P-1. The resultant constructed pGEX-RV-M plasmid was transformed into BL21 (DE3) and protein expression was analyzed by SDS-PAGE and Western blot. The results of SDS-PAGE showed that the M protein was efficiently expressed, which were mainly soluble, and purified with the affinity chromatography. The results of Western blot indicated that the recombinant protein M showed good immunogenicity. The indirect ELISA method was established with the purified recombinant protein M, and a total of 95 serum samples were detected by the method and the indirect ELISA method based on the recombinant protein P respectively. The results showed that compared with the commercially available ELISA kit coating RV as antigen, the indirect ELISA method based on recombinant protein P had a higher coincidence rate, and it can replace the ELISA method based on RV.

 The objective of this study was to obtain functional genes of Babesia motasi, a cDNA expression library of the merozoites was constructed and immunoscreened with positive sera from sheep infected with B. motasi. The merozoites of B. motasi were purified from red blood cell with differential centrifugation. The mRNA was purified from extracted total RNA. Synthetized double-strand cDNA was added directional EcoRⅠ/Hind Ⅲ linkers and ligated to the EcoRⅠ/HindⅢ arms of λ screen vector. To produce a primary cDNA library of B. motasi, the phages DNA was packaged in vitro and transfected into ER1647. The positive clones were obtained by immunoscreening with the positive sera against B. motasi and amplified with rapid amplification of cDNA ends (RACE). The titers of the primary and amplified cDNA expression library were 1.0×10⁶ PFU and 3.5×10⁹ PFU·mL^-1, respectively. The results showed that 10 genes of B. motasi were identified and the full-length of 8 genes were amplified by RACE. The cDNA expression library and genes screened provide an important material for screening and identifying candidate antigens of vaccination and diagnosis, drug targets as well studying biological characteristics of Babesia.

The objective of this study was to study the biological characteristics of goose AvBD1 and preliminary attempts to explore its resistance to Salmonella enteritidis mechanism. The mRNA of goose AvBD1 was cloned from bone marrow of the gooses by RT-PCR. The cDNA of goose AvBD1 was sub-cloned into EcoRⅠ and SalⅠ sites of pGEX-6p-1 vector to construct recombinant plasmid pGEX-goose AvBD1. The recombinant plasmid was transformed into E. coli BL21 and the bacteria was induced with IPTG. It was demonstrated by SDS-PAGE that a 31 kDa protein which was equal to goose AvBD1 protein in molecular weight was highly expressed. The sequence analysis showed that the gene fragment of AvBD1 contained 198 bp, and encoded 65 amino acids. Homology analysis showed that goose AvBD1 shared the highest percentage of amino acid homology (77.1%) with ostrich AvBD1.The purified recombinant goose AvBD1 exhibited extensive antimicrobial activity against twelve investigated bacteria strains, including Gram-positive and Gram-negative. At high salt ions conditions, antimicrobial activity of recombinant goose AvBD1 protein against both Staphylococcus aureus and Proteus mirabilis decreased significantly and hemolysis activity of the recombinant protein was extremely low. Experimental results show that Salmonella enteritidis did induced goose AvBD1 gene expression in bone marrow, at the same time goose AvBD1 resistance to Salmonella enteritidis infection induces expression of TLR4 may be mediated signal transduction.

The coding sequence of CD8α was cloned by RT-PCR and LD-PCR in five duck populations (Jinding Duck, Cherry Valley duck, muscovy, mallard and spot-billed duck). The 9 sequences （another 4 sequences from GenBank） were analyzed by online software of bioinformatics(the best nucleotide substitution model) was selected by Jmodeltest, the nucleotide substitution saturation was assessed with DAMBE, and the selective pressure on amino acid sites was tested by PAML. The results of sequencing showed the coding sequence of CD8α was conservative except a few point mutations. The model “GTR+G” was selected as the best nucleotide substitution model, the sequences were experienced full substitution unsaturation. The phylogenetic trees of CD8α among different fowl were generated by MEGA 5.0, and the 9 fowl were divided into 3 parts according to CD8α sequences. The 7 amino acid sites undergoing positive selection were detected, 4 of them were in the important function regions by the site-specific model. In summary, the coding sequence of CD8α was cloned successfully，and it was conservative in molecular evolution, which may be helpful for studying evolution, structure and function of CD8α among fowl.

 This experiment was conducted to study the change of the activity of NOS and the content of NO, and the influence of Xuesaitong and L-NNA on the change in Striatal Hemorrhage rabbits, and to investigate the mechanism of action of NO on the cerebral hemorrhage and the protective effect of those two pharmaceuticals on the cerebullar neurons. Fifty-six healthy Haerbin white rabbits, 4 moon's old, were divided into the model group, control group of sham operation and the treatment group of striatal hemorrhage rabbits with Xuesaitong and L-NNA injection in random. The mode of cerebral hemorrhage rabbits were established in this surgery , we determined the activity of NOS and the content of NO in different time after striatal hemorrhage in hemorrhagic corpus striatum by biochemical technology. The results indicated that the activity of NOS increased at 6 hours after striatal hemorrhage, and increased to the highest at 3 days, and then decreased gradually from 3 to 9 days, and basically returned to normal levels at 9 days. The activity of NOS in the two therapy groups were lower than that in model grope at every time extremely notable difference or notable difference (P<0.01 or P<0.05), and the level of decreasing the activity of NOS in the group of L-NNA was lower than that in the group of Xuesaitong between 6 hours to 1 day (P<0.05). The change of the content of NO and the activity of the NOS was accordant primitively and direct correlation. SABC immunohistochemical method was used to detected the neuronal NOS positive neurons in each corpus striatum. We found that the density of striatal neuronal NOS positive neurons increased, cell coloring became saturate, soma-sectional area and the length of longest tuber became smaller. The density of neuron reduced, and the soma-sectional area and the length of longest tuber ameliorated significantly after treatment (P<0.05). The results suggested that NO work in the process of cerebral hemorrhage damage, and the high concentrations of No play a neurotoxicity role in damage to brain tissue. Xuesaitong and L-NNA reduce the content of NO in the corpus striatum by inhibiting the activity of NOS, which has a significant protective effect on neurons in cerebral hemorrhage rabbits.

The aim of this study was to investigate the mediation mechanism of toll-like receptor 9 (TLR9) and the related inflammatory factors in brain injury. Immunohistochemical ultra sensitive SP method was used to examine the dynamic expression of TLR9 and the related factors in the normal rats, injected with E. coli and HuangLianJieDu Decoction preventive rats in the present study. 66 SD rats were randomly divided into three groups. The control group (n=6) was treated with normal saline by intraperitoneal injection. Experimental group (n= 30) was treated with E. coli culture by intraperitoneal injection 0.5 mL per rat (2.4×10⁹ CFU· mL^-1). HuangLianJieDu Decoction preventive groups (n=30) was treated with 12.6 mL·kg^-1orally in advance 6 days, and then were intraperitoneally injected with 0.5 mL (2.4×10⁹ CFU· mL^-1) per rat E. coli culture. Each group was divided into 6 sub-groups and each of which has 5 rats. The rats of each experimental group, prevention group and one rat in control group were killed randomly at 3, 6, 9, 12, 18, 24 hours after treated with E. coli. And then the expression of TLR9 and the related factors in brain were analyzed by immunohistochemistry. The results showed that there was a little amount of TLR9 expression in the brain under normal circumstances, while the TLR9 expression showed a significant change after E. coli infection, and it was more obvious over time. Compared with control group and prevention group, the expression of TLR9 increased significantly after 3 hours infected with E. coli, and it reached the peak after 12 hours (P＜0.01). There was no significant difference between prevention and control group (P＞0.05). These results indicated that the HuangLianJieDu Decoction can protect the brain injury by bacterial infection by suppressing the expression of TLR9.

In order to study clinical safety of Shegan Dilong particles, the safety pharmacology study was carried out for the first time by non-invasive way. Three doses of Shegan Dilong particles including high dose (2.0 g·kg^－1), medium dose (1.0 g·kg^－1) and low dose (0.5 g·kg^－1, which is equivalent to the recommended dose) were given to the anesthetized dogs, and there was also a group of anesthetized dogs given saline. Then the mean arterial pressure, heart rate, body temperature, standard Ⅱ lead ECG, respiratory rate, tidal volume, oxygen saturation, respiration curve, urine of eleven indicators, urine weight gain and other related indicators of the anesthetized dogs were observed. Meanwhile Kunming strain mice were given the particles in accordance with the above different doses, and the spontaneous activities of mice were observed. The above indicators observed were used to illustrate the impact of the particles on the cardiovascular system, respiratory system, urinary system and central nervous system. The results showed that Shegan Dilong particles given by oral according to the dose of 2.0 g·kg^－1 had no significant effect on the cardiovascular system, respiratory system and urinary system of anesthetized dogs. The results of the mice’s spontaneous activities also showed that the total distance, the side of distance, the angle of distance and time among the groups had no significant differences (P>0.05). And the general conduct of activities, posture and gait of the mice performed normal, while muscle trembling, anxiety, running, screams and other abnormalities in mice didn’t exit. It indicated that in the range of the test doses, the particles had no effect on the central nervous system in mice. The results suggested that at least in the range of 0.5-2.0 g·kg^－1 the Shegan Dilong particles had no significant effect on animal cardiovascular system, respiratory system, urinary system and central nervous system, and it also suggested that the adverse reactions was small. So it’s a safe drug suitable for the clinical application.

To investigate the spatial and temporal expression profiles of HSP90 gene in fibroblasts of the Northeast Wild boar under cold stress. Real-time fluorescent quantification reverse transcriptase PCR (FQ-RT-PCR) was applied to analyze the expression of HSP90 mRNA in the Northeast Wild boar fibroblasts under 4,15,25 and 32 ℃. The results showed that HSP90 mRNA transcription level didn’t significantly increase (P>0.05) under cold stress treatments at different temperatures; During rewarming culture, HSP90 mRNA transcription levels increased significantly (P<0.05) within 8 h of rewarming incubation following preincubation at 4 or 15 ℃ and the peak showed at the 6^th hour. While HSP90 mRNA transcription levels didn’t increase significantly in the 25 or 32 ℃ preincubation groups within 8 h of rewarming incubation; HSP90 mRNA transcription levels increased with decreasing of the temperature and duration of the cold treatment time when cells were preincubated at 4 or 15 ℃ for 2, 4, 6 or 8 h followed by a 4 h rewarming incubation. However, rewarming incubation didn’t significantly induce transcription of HSP90 mRNA in cells preincubated at 25 or 32 ℃ for 2, 4, 6 or 8 h. The result indicated that cold stress induced increase of HSP90 mRNA transcription level in fibroblasts of the Northeast Wild boar，did not occur in the stress period during low temperature treatment, but in the cellular stress period after rewarming. Mild cold stress (25-32 ℃) didn’t induce a significant increase at HSP90 mRNA transcript levels，while harsh cold stress (4-15 ℃) induced a significant increase at HSP90 mRNA transcript levels which were directly proportional to the intensity and duration of cold exposure at 4-15 ℃.

The objective of the experiment was to study the localition and the average integrated optical density of ERα and PR immunopositive cells and the abundance of ERα mRNA and PR mRNA in mammary gland during postnatal development in Jining Gray goat. The SP immunohistochemical staining and Image analysis methods were used to localize ERα and PR immunoprotective(-ip) cells in mammary gland of Jining Gray goat. The relative expression of ERα mRNA and PR mRNA was analyzed by using the real time fouorescence quantitative PCR with GAPDH as an internal gene. ERα and PR had similar variations, immunoprotective staining was mainly localized in the nucleus of blood vessels endothelial cells, stromal cells and ductal epithelial cells of mammary gland,occasionally in cytoplasmic, PR-ip substance also found in alveolar epithelial cells in 180 days.The average optical density was gradually increased from birth day to sexual maturity, and it was significantly higher in sexual maturity than that in birth day and puberty (P<0.01) ,it was not different with that in 180 days (P>0.05).The results of qRT-PCR showed that the abundance of ERα mRNA was gradually increased from birth day to sexual maturity , and it was significantly higher in sexual maturity than that in birth day and puberty (P<0.01),there was a slightly decrease from sexual maturity to 180 days .While the abundance of PR mRNA from birth day to sexual maturity was lower and there was no significant difference during these groups. In 180 days,it was significantly higher than the other three groups (P<0.01). These results indicated that the estrogen receptors of alph and the progesterone receptors could promote the growth of glandular ,the hyperplasia of epithelial cells and the development of lobulo-alveolar during postnatal development in Jining Gray goat.

The aim of this work is to seek a new rapid detection technology for Salmonella pullorum and Salmonella gallinarum. In this report, we firstly combined multiwalled carbon nanotubes with ionic liquid ［BMIM］PF₆ to modify four-channel screen-printed carbon electrode to propose an enzyme immunosensor with double amplified response signal for detection of Salmonella pullorum and Salmonella gallinarum. The surface morphology of the modified electrode was characterized by atomic force microscopy, while the electrochemical properties were determined by cyclic voltammetry. The results showed that under the optimal assay conditions, a good linear response occurred in the concentration range of 10³-10⁹ cfu·mL^－1 of Salmonella pullorum and Salmonella gallinarum, with a low detection limit of 3.93×10² cfu·mL^－1 (S/N=3). The stability test of the fabricated immunosensor showed that it remained 90.15% of its original response signal after storage of 28 days under 4 ℃. The performance evaluation of the proposed immunosensor showed that it had high stability, good specificity, acceptable reproducibility and accuracy. The results demonstrated that the proposed enzyme immunosensor with combination of multi-walled carbon nanotubes and ionic liquid achieved the effect of double amplification of detection signal which enabled it highly sensitive in detection. Ionic liquid was also helpful for maintaining bioactivity of enzyme labeled antibody, thereby prolonging effective time of the immune electrode. The fabrication of the enzyme immunosensor pursuant to this method provides a good reference model for construction of enzyme immunosensor for rapid detection of other pathogenic bacterium.

The aim of this study is to establish a multiplex real-time PCR assay to simultaneously detect and discriminate porcine reproductive and respiratory syndrome virus (PRRSV)，porcine circovirus type 2 (PCV2)，porcine pseudorabies virus（PRV）and porcine parvovirus (PPV) in a single test tube. According to the nucleotide sequences of PRRSV，PCV2, PRV and PPV from GenBank, four pairs of specific probes were designed. By optimizing the probe’s concentration, primers concentration and sample DNA extraction, a multiplex real-time quantitative PCR assay for detection of these viruses was established. The specificity and sensitivity of the multiplex real-time quantitative PCR assay were analyzed for single virus and multiple viruses. The results showed that the specificity was high, and no amplification was achieved to the other pathogeneses. Both multiplex and singleplex assays were consistently able to detect ＜10¹ copies of plasmid templates or 1 TCID₅₀ virus per reaction. 40 clinical samples were comparatively detected using the multiplex and singleplex assay. The data showed that the results of the singleplex and multiplex assay were in accordance with the results of conventional PCR. A multiplex real-time PCR assay was developed to simultaneously detect and discriminate PRRSV, PCV2, PRV and PPV in a single test tube, the method is rapid, sensitive, specific and accurate.