Significant association between IL-18 and OCT4 gene polymorphisms in susceptibility and clinical characteristics of prostate cancer

Objective Recent studies have shown abnormal expression of octamer-binding transcription factor 4(OCT4) and interleukin-18(IL-18) to be related to cancer. However, the molecular mechanisms by which the IL-18 and OCT4 gene polymorphisms are associated with prostate cancer remain unclear. In this study, we aimed to determine whether the presence of IL-18 and OCT4 polymorphisms were associated with size, grade, tumor, nodes and metastasis(TNM) stage, or survival in patients with prostate cancer.Methods Polymorphisms in OCT4 and IL-18 genes were evaluated to determine susceptibility to prostate cancer in 120 patients. A control group consisted of 125 Chinese participants. Genotyping was performed using TaqMan allelic discrimination assays, and statistical analysis was performed using SPSS. Results No association was found between OCT4 and IL-18 gene polymorphisms and prostate cancer susceptibility. For OCT4 AA and IL-18-607 CC genotypes, there was a significant association with higher tumor grade(P = 0.03 and P = 0.025) and stage(P = 0.04 and P = 0.001). The OCT4 and IL-18-137 GG genotype was correlated with higher tumor grade(P = 0.028) and stage(P = 0.008). Furthermore, OCT4 AA was significantly more frequent in patients with lymph node metastasis(P = 0.02) and distant metastasis(P = 0.01). The Cox proportional hazard model showed that tumor grade and stage grouping were independent prognostic factors but IL-18 and OCT4 polymorphisms were not. Conclusion The OCT4 gene may have a profound effect on prostate cancer risk. Polymorphism variants in the IL-18(IL-18-607 and IL-18-137) and OCT4 genes may be associated with poor prognoses for individuals with prostate cancer.

Single Nucleotide Polymorphisms rs2227284, rs2243283 and rs2243288 in the IL-4 Gene show no Association with Susceptibility to Chronic Hepatitis B in a Chinese Han Population

Objective To investigate the relationship between single nucleotide polymorphisms(SNPs) of the interleukin-4(IL-4) gene and outcome of hepatitis B virus(HBV) infection in a Chinese Han population.Methods Total of 501 patients with chronic hepatitis B virus(HBV) infection and 301 controls with selflimiting HBV infection were studied. Three tag SNPs in the IL-4 gene(rs2227284G/T, rs2243283C/G and rs2243288A/G) were genotyped by the Multiplex snapshot technique. The genotype and allele frequencies were calculated and analyzed.Results The three SNPs showed no significant genotype/allele associations with chronic HBV infection. Overall allele P values were: rs2227284, P = 0.655, odds ratio(OR) [95% confidence interval(CI)] = 1.070(0.793-1.445); rs2243283, P = 0.849, OR(95% CI) = 0.976(0.758-1.257); rs2243288, P = 0.659, OR(95% CI) = 1.060(0.818-1.375). Overall genotype P values were: rs2227284, P = 0.771; rs2243283, P = 0.571; rs2243288, P = 0.902. There were no statistically significant differences between patients with chronic HBV infection and controls. Haplotypes generated by these three SNPs also had no significant differences between the two groups. Conclusions The three tag SNPs of IL-4 were not associated with the outcome of HBV infection in the Han Chinese population.

Effect of remifentanil on toll-like receptor 4, NF-κB and IL-6 in rabbit myocardial ischemia/reperfusion model

Objective： To investigate whether remifentanil induced cardioprotecting effect is associated with expression of toll-like receptor 4 （TLR4）, nuclear factor rB （NF-r.B） and serum interleukin -6 （IL-6）. Methods： Fifty rabbits were randomly divided into 5 groups （n=10） according to the treatment： sham operation group （group A）, ischemla-reperfusion group （group B）, low-dose remifentanil group （group C）, mediate-dose remifentanil group （group D）, and high-dose remlfentanil group （group E） Myocardial TLR4 mRNA levels, NF-r.B protein expression and serum levels of IL-6 were observed in 120 min after reperfusion. Results： The myocardial expressions of TLR4 mRNA, NF-rd3 protein and IL-6 level in sera of groups B, C, D and E were elevated compared with group A. However, remifentanil significantly reduced the levels of TLR4 mRNA, NF- r.B protein expression and serum IL-6 in groups C, D and E compared with group B. There were remarkable differences between the groups （P〈O.O1）. Conclusion： Intravenous remifentanil has protective effect against rabbit myocardial ischemia/reperfusion injury. This effect may be associated with TLR4, NF-r.B expressions on myocytes and serum level of IL-6 in a dose-dependent manner

Impact of Yupingfengsan Oral Liquid on the Expression of IL-4, IFN-<i>γ</i>and NF-<i>κ</i>B of Allergic Rhinitis Mice

To observe the impact of Yupingfengsan Oral Liquid on the expression of IL-4 and IFN-γ in AR mice’s serum and expression level of nuclear factor—κB (NF-κB) protein and gene in nasal mucosa. Method: Forty BALB/c mice were randomly divided into the normal group, model group, Yupingfengsan Oral Liquid group (6 g/kg) and Loratadine group, with 10 mice per group. AR mice model was established by OVA, and IL-4 and IFN-γ contents can be measured with ELISA. The morphological changes of nasal mucosa were observed by hematoxylin eosin (HE) staining and NF-κB expression in the nasal mucosa of mice was tested with Real-Time PCR and Western blot. Results: Compared with the model group, the nasal symptoms in the Yupingfengsan Oral Liquid group and Loratadine group were obviously relieved. HE staining showed that there was a little inflammatory cell infiltration in the nasal mucosa of Yupingfengsan Oral Liquid group and Loratadine group and it was significantly reduced when compared with the model group. IL-4 level in the serum and expression of NF-κB protein and gene in the nasal mucosa was consistent and it was decreased when compared with the model group (P < 0.01), but the IFN-γ level in the serum was increased (P < 0.01). Conclusion: Yupingfengsan Oral Liquid can improve the clinical symptoms and histopathological manifestations of AR mice sensitized by OVA, inhibit the NF-κB expression, balance the percentage of Th1/Th2 cells, increase the IFN-γ level in the serum and decrease the IL-4 level.

Expression of toll-like receptor 4 gene polymorphism and inflammatory factors in peripheral blood on patients with sepsis

Objective:To observe the presence of toll-like receptor 4(TLR4)gene polymorphism in the venous blood in patients with sepsis,meanwhile,to observe the expression and the diagnostic value of TLR4 mRNA,interferon-(IFN-),interleukin-23(IL-23),procalcitonin(PCT)and C-reactive protein(CRP)in patients with different degrees of sepsis.Methods:The peripheral blood samples from the subjects were collected to extract genomic DNA,gene sequencing and enzyme digestion method were applied to the detection of TLR4 Asp299Gly loci polymorphism.The expression of TLR4 mRNA in the peripheral blood was detected by reverse transcriptase polymerase chain reaction(RT-PCR).The expression levels of IFN-,IL-23,PCT and CRP were measured by enzyme linked immunosorbent assay(ELISA).Results:(1)The gene polymorphism was not present in TLR4 Asp299Gly loci;(2)The expression of TLR4 mRNA in the peripheral blood in patients with sepsis:on d1,there were statistically significant differences between the normal group and the group(APACHE II≤20),between the normal group and the group(APACHE II>20),between the group(APACHE II≤20)and the group(AAPACHE II>20)(t=5.741,14.780 and 10.500,all p<.01).APACHE II stands for Acute Physiology and Chronic Health Evaluation II.On d7,there were statistically significant differences between the normal group and the group(APACHE II≤20),between the normal group and the group(APACHE II>20),between the group(APACHE II≤20)and the group(APACHE II>20)(t=4.186,13.830 and 9.645,all p<.01);(3)The expression levels of IFN-,IL-23,PCT and CRP were obviously upregulated(all p<.01),and TLR4 was positively correlated with IFN-and IL-23(all p<.01);(4)The best cutoff value of TLR4 mRNA at baseline was 891.6μg/L,with a sensitivity of 100%and a diagnostic specificity of 57%.The best cutoff value of IFN-at baseline was 84.5μg/L,with a sensitivity of 100%and a diagnostic specificity of 57%.The best cutoff value of IL-23 at baseline was 861μg/L,with a sensitivity of 100%and a diagnostic specificity of 97%.Conclusions:(1)The Asp299Gly polymorphism is not present in TLR4 gene;(2)The expression levels of TLR4 mRNA,IFN-and IL-23 are relatively high in patients with sepsis,and will be higher with the increased severity of sepsis;(3)TLR4 mRNA,IFN-and IL-23 can be used as molecular biomarkers for the early diagnosis of sepsis.

猪白细胞介素4的基因克隆及其在囊尾蚴DNA疫苗中的应用

目的:克隆猪白细胞介素4(IL-4)cDNA,观察其在抗囊尾蚴免疫中的疫苗佐剂作用。方法:通过RT-PCR法获得带有5'AUG侧翼翻译优化突变序列的猪IL-4 cDNA,测序后重组入真核表达载体pcDNA,在体外瞬时表达的基础上与囊尾蚴保护性抗原DNA疫苗联合免疫仔猪。结果:获得的猪IL-4 cDNA序列与文献报道的完全一致,在体外瞬时表达中获得了有生物学活性的猪IL-4。其与囊尾蚴保护性抗原DNA疫苗联合应用可有效提高个体体液免疫应答水平。结论:构建了一高效表达猪IL-4的真核表达质粒,初步实验证实了其在抗囊尾蚴DNA疫苗中的佐剂效应。

猪白细胞介素-4基因的克隆与表达

利用RT-PCR技术,从被刺激诱导的PBMC中克隆IL-4基因,序列分析表明:克隆的猪IL-4基因序列与Gen-Bank上登录的IL-4基因的核苷酸和氨基酸序列同源性分别为99%和97.8%。然后用表达型引物从T载体上扩增IL-4基因,双酶切PCR产物后与表达载体pGEX连接,构建重组表达质粒pGEX-IL-4,用IPTG诱导表达,表达产物经SDS-PAGE分析表明,表达出38 ku融合蛋白,占菌体总蛋白的30%以上,并且以包涵体的形式存在,这为猪重组IL-4规模化生产和疫苗佐剂的研制奠定了基础。

猪白细胞介素4,6融合基因和CpG基序对猪蓝耳病疫苗免疫的强化效应

为探索新型高效安全的猪蓝耳病疫苗免疫佐剂,本实验用离子交联法制备壳聚糖纳米颗粒包裹猪融合基因PIL-46和CpG基序的真核表达质粒(CNP-(VRIL-46+VR1C)),将CNP-(VRIL-46+VR1C)肌注接种过猪蓝耳病灭活苗的长白川白杂交猪,接种后1、2、4、6和8周采取实验猪静脉血,用Sandwich ELISA检测血清中白细胞介素2(IL-2)、IL-4、IL-6和IgG、IgA、IgM及特异抗体的含量,同时检测外周血液免疫细胞数量的变化。结果发现实验猪接种CNP-(VRIL-46+VR1C)后1～8周内,其血清中IL-2、IL-4、IL-6和IgG、IgA、IgM及特异抗体的含量较对照组均显著增多(P<0.05),淋巴细胞和单核细胞数量也明显升高(P<0.05)。这些表明接种CNP-(VRIL-46+VR1C)的实验猪的特异体液和细胞免疫水平均显著多于对照组,提示PIL-46基因和CpG基序组合能显著增强猪的特异体液和细胞免疫机能,明显提高其免疫防御力,具有研制为高效安全的分子免疫增强剂的应用前景。

表达PEDV中国变异株S蛋白复制型重组人腺病毒4型的构建及其免疫原性研究

为构建表达猪流行性腹泻病毒(PEDV)S蛋白复制型重组人腺病毒4型(HAdV-4)及评价其免疫原性,本研究利用RT-PCR扩增得到PEDV中国变异株纤突(S)基因的N-末端区域(1 bp^1 140 bp),构建重组质粒p Ad4FAST-Shuttle-S,重组质粒经线性化和同源重组,得到含有PEDV S基因的重组HAdV-4感染性克隆,纯化的感染性克隆DNA经PacⅠ线性化后转染HEK293细胞,拯救得到表达PEDV变异株S蛋白的重组病毒rAd4△E3-S。将该重组病毒免疫小鼠,间接ELISA测定其血清中抗S蛋白抗体水平,利用体外淋巴细胞转化试验和流式细胞技术检测免疫小鼠T淋巴细胞反应。结果显示,重组病毒能够表达PEDV S蛋白,并且能刺激小鼠产生特异性体液和细胞免疫应答。本实验结果为进一步研究重组病毒在猪体上的免疫反应奠定基础,也为PEDV活载体疫苗的研发提供实验依据。

孟鲁司特联合沙美特罗替卡松对咳嗽变异性哮喘患者白介素-4和γ-干扰素水平的影响

目的探讨孟鲁司特联合沙美特罗替卡松对咳嗽变异性哮喘患者白介素-4(IL-4)和γ-干扰素(IFN-γ)水平的影响。方法选择我院收治的咳嗽变异性哮喘患者74例,随机分为实验组和对照组。两组患者均予以沙美特罗替卡松吸入,1吸/次,2次/d。实验组在此基础上口服孟鲁司特咀嚼片10 mg,1次/d,连用12周。观察两组患者治疗前后血清IL-4和IFN-γ水平的变化,并观察临床疗效及安全性。结果治疗12周后,两组患者血清IL-4水平较治疗前明显下降,IFN-γ水平较治疗前明显上升(P<0.01或P<0.05),且实验组下降或上升值明显大于对照组(P<0.05);同时实验组的临床总有效率明显高于对照组(χ2=4.16,P<0.05)。对照组和实验组治疗过程中分别出现不良反应3例和6例,症状较轻,未发生严重药物不良反应。两组治疗过程中不良反应发生率比较差异无统计学意义(χ2=0.51,P>0.05)。结论孟鲁司特联合沙美特罗替卡松治疗咳嗽变异性哮喘效果明确,能降低血清IL-4水平,提高血清IFN-γ水平,纠正Thl/Th2细胞因子平衡失调,且安全性较高。

野猪源猪圆环病毒4型的发现及其Cap基因序列分析

猪圆环病毒4型(PCV4)于2019年在我国家猪中首次检测到,并发现其可能与多系统呼吸道疾病、腹泻及皮炎肾病综合征疾病有关。为了解该病毒在野猪中的流行和遗传进化特征,本研究采集江西省山区野猪组织样品25份,包含淋巴结、肾脏、脾脏,采用探针法荧光定量PCR(Taq Man qPCR)对组织样品检测,对PCV4阳性组织样品采用普通PCR扩增其Cap基因并分析其同源性及遗传进化关系。结果显示,25份样品中检测到3份PCV4阳性样品,检出率为12%(3/25);普通PCR扩增结果显示,在3份阳性样品中均扩增到约1300 bp的目的条带,测序后的序列比对分析结果显示其均为PCV4全长Cap基因序列(JXNC-2020、JXYC-2020及JXGZ-2020)。Cap基因的同源性分析显示,3个野猪源PCV4 Cap基因序列间的同源性为98.69%~100%,氨基酸序列间的同源性为97.1%~100%;与韩国野猪源PCV4流行株E115 Cap基因序列的同源性为98.11%~98.54%,氨基酸序列的同源性为96.93%~98.25%;与国内家猪源PCV4 Cap基因序列的同源性为98.54%~99.13%,氨基酸序列同源性97.37%~99.12%;与E115株相比,本研究获得的3个PCV4 Cap蛋白氨基酸序列分别存在3个及7个突变氨基酸。与国内PCV4相比,PCV4 Cap基因JXGZ-2020编码的氨基酸序列突变数明显少于其与韩国流行株相比。Cap基因的系统发育分析显示,野猪源PCV4与家猪源PCV4同属于一个分支,与圆环病毒属其他病毒处于不同分支。上述结果表明,野猪源PCV4与家猪源PCV4的亲缘关系较近,间接表明野猪可以促进PCV4的扩散与传播。本研究结果首次拓展了PCV4的宿主范围,为进一步研究PCV4的流行及基因组特征提供参考。

Gene polymorphisms of interleukin-28, p21-activated protein kinases 4, and response to interferon-α based therapy in Chinese patients with chronic hepatitis B

Background Peg-lnterferon-a treatment is expensive and associated with considerable adverse effects, selection of patients with the highest probability of response is essential for clinical practice. The objective of this study was to assess the relationship between the gene polymorphisms of interleukin-28 （IL-28）, p21-activated protein kinase 4 （PAK4） and the response to interferon treatment in chronic hepatitis B patients. Methods Two hundred and forty interferon-naive treatment HBeAg seropositive chronic hepatitis B patients were enrolled in the present prospective nested case-control study. Peripheral blood samples were collected, including 92 with favorable response and 148 without response to the interferon treatment. Rs8099917, rs12980602, and rs9676717 SNP was genotyped using matrix assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF MS）. Results IL-28 genotype was not associated with response to interferon treatment （OR for GT/GG vs. TT, 0.881 （95% CI 0.388-2.002）; P=0.762; OR for CT/CC vs. TT, 0.902 （95% CI 0.458-1.778）; P=-0.766）. Rs9676717 in PAK4 genotype was independently associated with the response （OR for CT/CC vs. TT, 0.524 （95% CI 0.310-0.888）; P=0.016）. When adjusting for age, gender, smoking, drinking, levels of hepatitis B virus DNA, and alanine aminotransferase （ALT）, rs9676717 genotype TT appeared to be associated with a higher probability of response for interferon treatment （OR, 0.155 （95% CI 0.034-0.700）; P=0.015）. Conclusion Genotype TTfor rs9676717 in PAK4 gene and no drinking may be predictive of the interferon-a treatment success.

Association between the polymorphisms of interleukin-4, the interleukin-4 receptor gene and asthma

Background Interleukin-4 （IL4） is one of the most important cytokines involved in a variety of allergic disorders, particularly, asthma. A number of genetic epidemiological studies have identified an association between the gene polymorphisms of IL4 and interleukin-4 receptor （IL4R） and asthma in different populations. However, these studies have been inconsistent and inconclusive. The aim of this study was to investigate the association between the single nucleotide polymorphism （SNP） of IL-4, IL-4R and asthma risk in case-controlled studies using meta-analysis. Method A genetic model-free approach was used to perform the meta-analysis. Asthma （atopy status nondefined）, nonatopic and atopic asthma subgroups were separately analyzed. Next, the ethnic subgroup was analyzed. Heterogeneity and publication bias were also explored. Results Only two polymorphisms of IL4 （rs2243250 and rs2070874） and four polymorphisms of IL4R （rs1801275, rs1805011, rs1805010, and rs1805015） were included in the meta-analysis. Polymorphisms rs2243250 and rs2070874 of IL-4 and rs1801275 and rs1805011 of IL4R were associated with asthma. The overall odds ratio （OR） of rs2243250 in the CC versus TT＋TC genotypes was 0.84 （95% CI： 0.75-0.94）, and the Z-test for the overall effect was 3.0 （P=0.003）. We obtained significant results from this polymorphism in the Caucasian ethnicity and adult groups. However, the overall OR of rs1801275 for the GG＋AG versus AA genotype was 1.16 （95% CI： 1.00-1.35）, and the Z-test for the overall effect was 1.87 （P=0.06）. Moreover, significant results were only obtained from the sub-group analysis in Asians （P=0.02）. In the rs1805011 polymorphism of IL4R, the overall OR for the CC ＋AC versus AA genotypes was 0.39 （95% CI： 0.16-0.95）, and the Z-test for the overall effect was 2.08 （P=0.04）. Conclusions Both the IL4 and IL4R polymorphisms were associated with asthma. The rs2243250 polymorphism of IL4 was more important in the white and adult groups. Individuals who carried the C allele for rs2070874 of the IL4 gene demonstrated increased asthma risk compared to TT homozygotes. An individual with an AA genotype in rs1805011 of the IL4R gene was less likely to suffer from asthma compared to the other two genotypes.

Immune Responses to Trichloroethylene and Skin Gene Expression Profiles in Sprague Dawley Rats

Objective To characterize the immune reaction in SD rats exposed to trichloroethylene （TCE） and to identify the gene expression profiles involved in skin after TCE exposure. Methods Fifteen percent of TCE was injected intradermally into the rat back （100 μL/120 g） at intervals of 7 days. Whole blood was collected 24 h after the fifth or seventh intradermic administration of TCE. The percentages of CD4＋ and CD8＋ of T lymphocytes were measured by a flow cytometer. The concentrations of IFN-gamma and 1L-4 in the serum were semi-quantified by ELISA. Total RNAs of skin samples at 3 h or 24 h after the seventh dose of TCE in SD rats were extracted, and gene expression proftles of these tissues were analyszed by rat toxicology U34 array of Affymetrix. Results Obvious decline of CD4＋ in T lymphocytes was observed in the TCE-administer group. No significant concentration differences in IFN-gamma and IL-4 were found between TCE-treated and control rats. Gadd45a and Mel were significantly up regulated in skin tissue 24 h after TCE exposure. The expression regulation of immune response factors was as active as proteins associated with lipid metabolism and synthesis process in these skin samples of SD rats exposed to TCE. Conclusion T-helper type 1 cells mediate immune response can not be elicited in TCE-treated SD rats, but certain immune disorder can be induced.

猪圆环病毒4型实时荧光RAA检测方法的建立

为快速检测猪圆环病毒4型(PCV4),本研究根据PCV4的Cap基因片段保守区设计引物和探针,建立了PCV4重组酶介导核酸等温扩增荧光法(RAA),并应用该方法对40份猪组织样品进行检测。结果表明,该方法可在20 min内,42℃恒温条件下特异性检测出PCV4,以猪圆环病毒2型(PCV2)、猪圆环病毒3型(PCV3)、猪流行性腹泻病毒(PEDV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪传染性胃肠炎病毒(TGEV)等病毒核酸为模板进行反应的扩增结果均为阴性;最低检出限至7.42×10^(1)拷贝/μL,灵敏度较高。利用建立的RAA检测方法和常规PCR法分别对猪组织样本进行检测,均未检出,表明当前猪场流行率较低。综上所述,本研究建立的PCV4实时荧光RAA检测方法快速简便、特异性强、灵敏度较高,可应用于PCV4的临床筛查与流行病学研究。

Retrovirus-mediated delivery of an IL-4 receptor antagonist inhibits allergic responses in a murine model of asthma

This work reports the investigation of the effect of airway IL-4RA gene transfer by a recombinant retroviral vector on airway inflammation and airway responsiveness in asthmatic mice. The retrovirus-mediated delivery of IL-4RA to the airways of mice inhibited elevations of airway responsiveness and the development of allergic inflammation in asthmatic mice, and regulated the Th1/Th2 balance in OVA-sensitized and -challenged mouse models. This suggests that gene therapy is a therapeutic option for treating and controlling chronic airway inflammation and asthma symptoms.

Effects of Electroacupuncture at Shangjuxu (ST 37) on Interleukin-1β and Interleukin-4 in the Ulcerative Colitis Model Rats

Objective： To probe into the mechanisms of electroacupuncture （EA） at Shangjuxu （ST 37） for treatment of the ulcerative colitis （UC）. Methods： Forty male Wistar rats were randomly divided into 4 groups： a normal group, a model group, a Shangjuxu group and a non-acupoint group, 10 rats in each group. The UC rat model was made with enema of trinitro-benzenesulfonic acid （TNBSA）, and the changes of interleuldn-1β （IL-1β） and interleukin-4 （IL-4） contents after EA at Shangjuxu （ST 37） were observed. Results： EA at Shangjuxu （ST 37） could significantly decrease the IL-1β content and increase the IL-4 content in the colic tissues of the UC rats with significant differences as compared with the model group and the non-acupoint group （P〈0.05 or P〈0.01）. Conclusion： The mechanisms of EA at Shangjuxu （ST 37） for treatment of the UC rats is possibly related with the decrease of IL-1β, a inflammation-promoting cytokine, and the increase of IL-4, a anti-inflammatory cytokine.

猪三种细胞因子基因的克隆与序列测定

利用RTPCR技术,从植物凝集素(PHA)、脂多糖(LPS)诱导的猪外周血淋巴细胞(PBMC)中成功地克隆了IL2、IL4、IFNα1基因,诱导后第6～24h细胞因子转录基因量最丰富;诱导剂LPS和PHA联合使用的效果优于LPS或PHA单独使用的诱导效果。序列测定与分析表明,克隆的IL2、IL4和IFNα1基因与相应的参考序列的同源性分别是98.5%、99.0%和97.6%。

猪圆环病毒3型和4型双重PCR检测方法的建立

为了检测和鉴别猪圆环病毒3型(PCV3)和4型(PCV4),根据GenBank已登录的PCV3和PCV4基因序列,在PCV3 Rep和PCV4 Cap基因的高度保守区域设计并合成2对特异性引物,经过双重PCR反应条件优化,建立了能够同时检测PCV3/4的双重PCR方法。结果显示,该方法能同时扩增出138(PCV3)和391 bp(PCV4)特异片段,而对PCV2、猪伪狂犬病病毒、猪细小病毒、猪繁殖与呼吸综合征病毒、猪瘟病毒和猪流行性腹泻病毒核酸扩增结果均为阴性。PCV3/4的检测极限分别为74.5,90.2拷贝/μL。经临床样本检测结果表明,本试验建立了同时检测PCV3/4双重PCR方法,且具有准确、可靠、灵敏度高、特异性强的特点,为PCV3/4的临床检测和鉴别诊断提供了技术支持。

检测猪圆环病毒4型实时荧光PCR方法的建立及应用

参照GenBank收录的PCV4 ORF2基因保守序列,设计合成了1对特异性引物和TaqMan探针,通过优化反应体系,建立了针对PCV4的实时荧光PCR方法,并对该方法的特异性、灵敏性和重复性进行评价。结果显示,该方法的Ct值与标准品在1.53×10^(3)~1.53×10^(7)拷贝/μL范围内呈良好的线性关系,R^(2)值为0.999;特异性强,仅对PCV4呈特异性扩增,与PCV1、PCV2、PCV3以及其他多种猪常见病原均无交叉反应;敏感性高,最低检测限为1.53×10^(1)拷贝/μL反应;重复性好,组内、组间变异系数均小于2%。使用该方法对采集自2013—2020年间河北省10个地区不同养殖场和屠宰场的868份猪组织样本进行PCV4检测,结果表明PCV4的阳性检出率为1.27%(11/868)。ORF2基因测序分析表明,河北省分离株同国内其他分离株的ORF2同源性为98.4%~99.6%。本研究建立的实时荧光PCR方法对PCV4的病原学监测和流行病学调查具有重要意义。