Novel methylation specific real-time PCR test for the diagnosis of Klinefelter syndrome

The aim of this study was to design a molecular assay for the diagnosis of Klinefelter syndrome （KS）, based on the detection of supernumerary X-chromosomes （X-chs）. DNA was extracted from peripheral blood samples of twenty-six 47,XXY males; two 46,XY/47,XXY males; twenty-two 46,XY males; and 15 females; and deaminated. Methylation-specific quantitative polymerase chain reaction （MS-qPCR） was performed using primers for unmethylated and methylated copies of the X-ch inactive-specific transcript （XIST-U and XIST-M） gene. X-ch disomy was determined on the basis of XIST methylation status. Degree of mosaicism in the 46,XY/47,XXY males was compared with karyotype and fluorescent in situ hybridization （FISH） results. Data analysis was performed using the Roche LightCycler software V. 3.5.3, including determination of crossing points （CPs） by fit-point analysis and melting curve analysis. Xoch disomy was detected in all female controls and KS patients; male controls expressed XIST-M only. CPs ranged from 29.5 to 32.5 （standard deviation （s.d.） 0.8） for XIST-U and from 29 to 31 （s.d. 0.6） for XIST-M. Limit of detection of mosaicism was 1%. Based on XlST-U/XIST-M ratios for the two 47,XXY/46,XY patients, the calculated degree of mosaicism （1.8% and 17.8%） was comparable to FISH results （2.3% and 15%, respectively）. Turnaround time from DNA deamination to final data analysis was under 9 h. We conclude that MS-qPCR is a sensitive, specific and rapid test for the detection of X-ch disomy, with applicability for the screening and diagnosis of KS, even in the setting of low grade 47,XXY/46,XY mosaicism.

Droplet digital polymerase chain reaction assay for methylated ring finger protein 180 in gastric cancer

BACKGROUND Gastric cancer(GC)is one of the most prevalent malignant tumors that endangers human health.Early diagnosis is essential for improving the prognosis and survival rate of GC patients.Ring finger protein 180(RNF180)is involved in the regulation of cell differentiation,proliferation,apoptosis,and tumorigenesis,and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC.Thus,methylated RNF180 can be used as a potential biomarker for GC diagnosis.AIM To use droplet digital polymerase chain reaction(ddPCR)to quantify the methylation level of the RN180 gene.A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.METHODS The primer and probe were designed and selected,the conversion time of bisulfite was optimized,the ddPCR system was adjusted by primer concentration,amplification temperature and amplification cycles,and the detection limit of ddPCR was determined.RESULTS The best conversion time for blood DNA was 2 h 10 min,and that for plasma DNA was 2 h 10 min and 2 h 30 min.The results of ddPCR were better when the amplification temperature was 56°C and the number of amplification cycles was 50.Primer concentrations showed little effect on the assay outcome.Therefore,the primer concentration could be adjusted according to the reaction system and DNA input.The assay required at least 0.1 ng of input DNA.CONCLUSION In summary,a ddPCR assay was established to detect methylated RNF180,which is expected to be a new diagnostic biomarker for GC.

Comparison of direct fecal smear microscopy,culture,and polymerase chain reaction for the detection of Blastocystis sp.in human stool samples

Objective:To compare the sensitivity and specificity of direct fecal smear microscopy,culture,and polymerase chain reaction in the detection of Blastocystis sp.in human stool.Methods:Human stool samples were collected from a community in San Isidro,Rodriguez,Rizal,Philippines.These samples were subjected to direct fecal smear microscopy,culture and polymerase chain reaction to detect the presence of Blastocystis sp.Results:Of the 110 stool samples collected,28(25%)were detected positive for the presence of Blastocystis sp.by two or more tests.Culture method detected the highest number of Blastocystis-positive stool samples(n=36),followed by PCR of DNA extracted from culture(n=26),PCR of DNA extracted from stool(n=10),and direct fecal smear(n=9).Compared to culture,the sensitivity of the other detection methods were 66.7%for PCR from culture and 19.4%for both PCR from stool and direct fecal smear.Specificity of the methods was high,with PCR from culture and direct fecal smear having97.3%,while PCR from stool at 95.9%.Conclusions:In this study,in vitro culture is the best method for detecting Blastocystis sp.in human stool samples.

马乳酒样乳杆菌马乳酒样亚种real-time PCR检测方法的建立与应用

本研究建立了一种特异性实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)检测方法,根据模式菌株马乳酒样乳杆菌马乳酒样亚种ZW3的16S rDNA序列和全基因组序列设计筛选特异性引物,采用SYBR Green I荧光染料建立real-time PCR方法,并对方法的特异性、灵敏度、重复性和混合体系等进行检测。结果表明,本研究所建立的方法特异性强、灵敏度高、重复性好,建立real-time PCR的标准曲线,其决定系数R2为0.965,具有良好的线性关系,且在马乳酒样乳杆菌马乳酒样亚种及混合体系中可以特异性检出。综上,本研究建立的real-time PCR法可以快速、准确地检测马乳酒样乳杆菌马乳酒样亚种,为马乳酒样乳杆菌的特异性定性定量检测提供了一种新的方法。

MGMT is down-regulated independently of promoter DNA methylation in rats with all-trans retinoic acidinduced spina bifida aperta

O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expression and methylation levels in the early embryo and in different embryonic stages, as well as the relationship between MGMT and neural tube defects. Spina bifida aperta was induced in rats by a single intragastric administration of all-trans retinoic acid on embryonic day(E) 10, whereas normal control rats received the same amount of olive oil on the same embryonic day. DNA damage was assessed by detecting γ-H2 A.X in spina bifida aperta rats. Real time-polymerase chain reaction was used to examine mRNA expression of MGMT in normal control and spina bifida aperta rats. In normal controls, the MGMT mRNA expression decreased with increasing embryonic days, and was remarkably reduced from E11 to E14, reaching a minimum at E18. In the spina bifida aperta model, γ-H2 A.X protein expression was increased, and mRNA expression of MGMT was markedly decreased on E14, E16, and E18. Bisulfite sequencing polymerase chain reaction for MGMT promoter methylation demonstrated that almost all CpG sites in the MGMT promoter remained unmethylated in both spina bifida aperta rats and normal controls, and there was no significant difference in methylation level between the two groups on either E14 or E18. Our results show that DNA damage occurs in spina bifida aperta rats. The mRNA expression of MGMT is downregulated, and this downregulation is independent of promoter DNA methylation.

Regeneration and DNA demethylation do not trigger PDX-1 expression in rat hepatocytes

AIM:To explore the possibility that PDX-1 gene is reactivated as a consequence of molecular events that occur during liver regeneration. METHODS:Rat hepatocytes were maintained in DMEM- F12,10%fetal bovine serum(FBS),penicillin/streptomycin and geneticin when applicable.Rat insulinoma RIN 1046-38 cells were maintained in M-199-10%FBS and penicillin/streptomycin.The final concentration of glucose was 11.1 mmol/L.During regeneration,lateral and medial liver lobes of adult male Wistar rats were surgically removed,with up 70%loss of liver mass.In methylation experiments,5-aza-deoxycytidine(5-aza-dC)was used.Primer3 software was used for poly- merase chain reaction(PCR).Quantitative real time PCR (qRT-PCR)was performed using SYBR Green technol- ogy;primers were designed by Beacon Designer 6 software.Western blotting and SDS-PAGE were performed according to standard procedures.Antibodies were purchased from commercial suppliers.RESULTS:We explored the possibility that liver regeneration could trigger PDX-1 expression,and hence insulin production.Twenty-four hours after surgical liver removal,regeneration was active as demonstrated by the increased proliferating cell nuclear antigen;however, all the other checked genes(involved in insulin gene expression):PC-1,Ngn3,NeuroD1,Btc,PDX-1 and Ins-1, were not related to the molecular events caused by this process.The only marker detected in regenerating liver was E47:a transcription factor of the the basic helixloop-helix family known to be expressed ubiquitously in mammalian cells.In the rat pancreas,almost all of the tested genes were expressed as shown by RT-PCR, except for Ngn3,which was silenced 2 d after birth. Therefore,the molecular events in liver regeneration are not sufficient to promote PDX-1 expression.DNA methylation is a known mechanism to achieve stable re- pression of gene expression in mammals:Hxk 2 gene is silenced through this mechanism in normal hepatocytes. The administration of 5-aza-dC to cultured cells is in fact able to upregulate Hxk 2 mRNA.We investigated whether PDX-1 silencing in liver cells could be exerted through methylation of CpG islands in both the promoter and the gene coding regions.The results show that the drug increased the expression level of the Hxk 2 control gene but failed to rescue the expression of PDX-1,thus DNA demethylation is not sufficient to override repression of the PDX-1 gene.

Demethylation of tumor necrosis factor-α converting enzyme promoter associated with high hepatitis B e antigen level in chronic hepatitis B

AIM: To evaluate tumor necrosis factor-α converting enzyme(TACE) methylation status in patients with chronic hepatitis B(CHB).METHODS: Eighty patients with hepatitis B e antigen(HBe Ag)-positive CHB, 80 with HBe Ag-negative CHB, and 40 healthy controls(HCs) were randomly enrolled in this study. Genomic DNA was extracted from peripheral blood mononuclear cells and methylation status of TACE promoter was determined by methylation-specific polymerase chain reaction. The clinical and laboratory parameters were collected.RESULTS: One hundred and thirty of 160 patients with CHB(81.25%) and 38 of 40 HCs(95%) displayed TACE promoter methylation. The difference was significant(χ2 = 4.501, P < 0.05). TACE promoter methylation frequency in HBe Ag-positive CHB(58/80, 72.5%) was significantly lower than that in HBe Ag-negative CHB(72/80, 90%; χ2 = 8.041, P < 0.01) and HCs(χ2 = 8.438, P < 0.01). However, no significant difference was observed in the methylation frequency between HBe Agnegative CHB and HCs(χ2 = 0.873, P > 0.05). In the HBe Ag-positive group, TACE methylation frequency was significantly negatively correlated with HBe Ag(r =-0.602, P < 0.01), alanine aminotransferase(r =-0.461, P < 0.01) and aspartate aminotransferase(r =-0.329, P < 0.01). CONCLUSION: Patients with HBe Ag-positive CHB have aberrant demethylation of the TACE promoter, which may potentially serve as a biomarker for HBe Ag seroconversion.

Aberrant gene methylation in the peritoneal fluid is a risk factor predicting peritoneal recurrence in gastric cancer

AIM:To investigate whether gene methylation in the peritoneal fluid (PF) predicts peritoneal recurrence in gastric cancer patients.METHODS: The gene methylation of CHFR (checkpoint with forkhead and ring finger domains), p16, RUNX3 (runt-related transcription factor 3), E-cadherin, hMLH1 (mutL homolog 1), ABCG2 (ATP-binding cassette, sub-family G, member 2) and BNIP3 (BCL2/adenovirus E1B 19 kDa interacting protein 3) were analyzed in 80 specimens of PF by quantitative methylation-specific polymerase chain reaction (PCR). Eighty patients were divided into 3 groups; Group A (n=35):the depth of cancer invasion was less than the muscularis propria; Group B (n=31): the depth of cancer invasion was beyond the muscularis propria. Both group A and B were diagnosed as no cancer cells in peritoneal cytology and histology; Group C (n=14): disseminated nodule was histologically diagnosed or cancer cells were cytologically defi ned in the peritoneal cavity.RESULTS: The positive rates of methylation in CHFR, E-cadherin and BNIP3 were significantly different among the 3 groups and increased in order of group A, B and C (0%,0% and 21% in CHFR, P<0.05; 20%, 45% and 50% in E-cadherin, P<0.05;26%,35% and 71% in BNIP3, P<0.05). In addition, the multigene methylation rate among CHFR, E-cadherin and BNIP3 was correlated with group A, B and C (9%,19% and 57%, P<0.001). Moreover, the prognosis was analyzed in group B, excluding 3 patients who underwent a non-curative resection. Two of the 5 patients with multigene methylation showed peritoneal recurrence after surgery, while those without or with a single gene methylation did not experience recurrence (P<0.05).CONCLUSION: This study suggested that gene methylation in the PF could detect occult neoplastic cells in the peritoneum and might be a risk factor for peritoneal metastasis.

p16 gene methylation in colorectal cancers associated with Duke's staging

AIM: To explore the association of methylation of the CpG island in the promotor of the p16 tumor suppressor gene with the clinicopathological characteristics of the colorectal cancers. METHODS: Methylation-specific PCR (MSP) was used to detect p16 methylation of 62 sporadic colorectal cancer specimens. RESULTS: p16 methylation was detected in 42% of the tumors.Dukes'staging was associated with p16 methylation status.p16 methylation occurred more frequently in Dukes'C and D patients (75.9%) than in Dukes'A and B patients (12.1%). CONCLUSION: p16 methylation plays a role in the carcinogenesis of a subset of colorectal cancer, and it might be linked to poor prognosis.

Study on RIZ1 gene promoter methylation status in human esophageal squamous cell carcinoma

AIM： To investigate the promoter region methylation status of retinoblastoma protein-interacting zinc finger gene 1 （RIZ1） in the human esophageal squamous cell carcinoma （ESCC） cell lines and tissues and verify the relationship between methylation of RIZ1 and oncogen- esis, tumor progression and metastasis etc of ESCC. METHODS： Methylation-specific polymerase chain reac- tion （MSP） was used to investigate the promoter region methylation status of RIZ1 in 6 ESCC cell lines. One cell line where RIZ1 promoter region methylation was de- tected was selected for the next study, where the cell line was treated with 5-aza-CdR. Real-time polymerase chain reaction was used to investigate its influence on the transcription of RIZ1. Experiments using frozenpathological specimens from 47 ESCC patients were performed using the same MSP methodology. RESULTS： Promoter methylation of RIZ1 gene was detected in TEl3, CaEs17 and EC109 cell lines and the cell line TEl3 was chosen for further study. The expression of RIZl mRNA in TE-13 was up-regulated after treatment with 5-aza-CdR. The rate of methyla- tion in carcinomas tissues was significantly higher than those in matched neighboring normal and distal ending normal tissue, and the deviation of data was statisti- cally significant （2,2 = 24.136, P 〈 0.01）. Analysis of the gender, age familial history, tumour deviation, tumour saturation, lymph gland displacement and clinical stag- ing of 47 samples from ESCC patients showed that the fluctuation of data was not statistically significant. CONCLUSION： Promoter methylation may play an im- portant role in the epigenetic silencing of RIZ1 gene expression in human ESCC. RIZ1 is considered to be a potential tumor suppressor gene and may be a biologi- cal parameter for testing early stage human ESCC.

Aberrant promoter methylation of p16 in colorectal adenocarcinoma in North Indian patients

AIM:To investigate p16 gene methylation and its expression in 30 patients with sporadic colorectal adenocarcinoma in a North Indian population. METHODS:Methylation specific polymerase chain reaction was used to detect p16 gene methylation and immunohistochemistry was used to study the p16 expression in 30 sporadic colorectal tumors as well as adjoining and normal tissue specimens.RESULTS:Aberrant promoter methylation of p16 gene was detected in 12(40%)tumor specimens,whereas no promoter methylation was observed in adjoining and normal tissue.Immunohistochemistry showed expression of p16 protein in 26(86.6%)colorectal tumors whereas complete loss of expression was seen in 4(13.3%)and reduced expression was observed in 12(40%)tumors. In the adjoining mucosa,expression of p16 was in 11 (36.6%)whereas no clear positivity for p16 protein was seen in normal tissue.There was a significant difference in the expression of p16 protein in tumor tissue and adjoining mucosa(P<0.001).The methylation of the p16 gene had a significant effect on the expression of p16 protein(P=0.021).There was a significant association of methylation of p16 gene with the tumor size (P=0.015)and of the loss/reduced expression of p16 protein with the proximal site of the tumor(P=0.047). Promoter methylation and expression of p16 had no relation with the survival of the patients(P>0.05). CONCLUSION:Our study demonstrated that promoter hypermethylation of the p16 gene results in loss/reduced expression of p16 protein and this loss/reduced expression may contribute to tumor enlargement.

Gamma-aminobutyric acid A receptor and N-methyl-D-aspartate receptor subunit expression in rat spiral ganglion neurons

BACKGROUND： Gamma-aminobutyric acid A （GABAA） and N-methyl-D-aspartate （NMDA） receptors are significant receptors in the central nervous system. An understanding of GABAA and NMDA receptor expression in spiral ganglion neurons （SGN） provides information for the functional role of these receptors in the auditory system. OBJECTIVE： To investigate mRNA expression of GABAA receptor （GABAAR） and NMDA receptor （NMDAR） subunits in the rat SGN. DESIGN, TIME AND SETTING： This in vitro, molecular biological study was performed at the Laboratory of Otolaryngology-Head and Neck Surgery, Guangxi Medical University, China from July 2007 to May 2008. MATERIALS： Reverse Transcriptase Kit and Taq DNA polymerase were purchased from Fermentas Burlington, ON, Canada; GABAAR and NMDAR primers were purchased from Shanghai Sangon, Shanghai, China. METHODS： SGN from 3-5 day postnatal Wistar rats was collected for primary cultures, mRNA expression of GABAAR and NMDAR subunits in the SGN was determined by reverse transcription polymerase chain reaction. MAIN OUTCOME MEASURES： Expression levels of GABAAR and NMDAR subunits were determined by quantitative analysis. RESULTS： GABAAR subunits （αl 6, β1 3, and y1 3） and NMDAR subunits （NR1, NR2A, NR2B, NR2C, NR2D, NR3A, and NR3B） were detected in the SGN. In α subunit genes of GABAAR, α1 and α3 expression was similar （P 〉 0.05） and greater than the other subunits. Of the β subunit genes, β1 subunit mRNA levels were greater than β2 and β3. Of the y subunit genes, y2 subunit mRNA levels were greater than y1 and y3. NR1 mRNA expression was the greatest of NMDAR subunits. CONCLUSION： GABAAR subunits （α1 6, β1-3, and y1-3） and NMDAR subunits （NR1, NR2A, NR2B, NR2C, NR2D, NR3A, and NR3B） were expressed in the rat SGN. Through comparison of GABAAR and NMDAR subunit expression, possible GABAAR combinations, as well as highly expressed subunit combinations, were estimated, which provided information for pharmacological and electrophysiological characteristics of GABAAR in the auditory system.

E-cadherin基因启动子区高甲基化与卵巢癌发生发展的相关性研究

目的探讨E-钙粘蛋白(E-cadherin)基因启动子区甲基化状态及其mRNA表达在卵巢癌发生发展中的作用。方法甲基化特异性聚合酶链反应(MSP)检测E-cadherin基因启动子区在48例卵巢癌组织及48例正常卵巢组织中的甲基化状态,荧光实时定量PCR(qRT-PCR)检测E-cadherin基因mRNA的表达情况。比较卵巢癌组织和正常卵巢组织中E-cadherin基因启动子区甲基化水平及其mRNA表达的差异情况,并分析它们与卵巢癌患者临床病理特征之间的关系。结果卵巢癌组织中E-cadherin基因启动子区甲基化阳性率显著高于正常卵巢组织(P<0.05),E-cadherin基因mRNA表达水平显著低于正常卵巢组织(P<0.05),且E-cadherin基因启动子区甲基化阳性的卵巢癌组织中E-cadherin mRNA表达量显著低于甲基化阴性的卵巢癌组织(P<0.05)。此外,卵巢癌组织中E-cadherin基因启动子区甲基化状态及其mRNA表达水平与卵巢癌患者的FIGO分期及淋巴结转移显著相关(P<0.05)。结论E-cadherin基因启动子区的高甲基化导致其表达下调可能在卵巢癌发生发展中起重要作用,E-cadherin基因甲基化状态有可能成为卵巢癌分子诊断和治疗的重要靶点。

RASSF1A methylation as a biomarker for detection of colorectal cancer and hepatocellular carcinoma

BACKGROUND Studies have validated the potential of methylated cell-free DNA as a biomarker in various tumors,and methylated DNA in plasma may be a potential biomarker for cancer.AIM To evaluate the diagnostic value of RASSF1A methylation in plasma for colorectal cancer(CRC)and hepatocellular carcinoma(HCC).METHODS A total of 92 CRC patients,67 colorectal polyp(CRP)patients,63 HCC patients,and 66 liver cirrhosis(LC)patients were enrolled.The plasma DNA was subjected to DNA extraction,double-strand DNA concentration determination,bisulfite conversion,purification,single-strand DNA concentration determination,and digital polymerase chain reaction(PCR)detection.The methylation rate was calculated.The diagnostic value was evaluated by the area under the curve(AUC).RESULTS The age and sex in the CRC and CRP groups and the HCC and LC groups were also matched.The DNA methylation rate of RASSF1A in plasma in the CRC group was 2.87±1.80,and that in the CRP group was 1.50±0.64.DNA methylation of RASSF1A in plasma showed a significant difference between the CRC and CRP groups.The AUC of RASSF1A methylation for discriminating the CRC and CRP groups was 0.82(0.76-0.88).The AUCs of T1,T2,T3 and T4 CRC and CRP were 0.83(0.72-0.95),0.87(0.78-0.95),0.86(0.77-0.95),and 0.75(0.64-0.85),respectively.The DNA methylation rate of RASSF1A in plasma in the HCC group was 4.45±2.93,and that in the LC group was 2.46±2.07.DNA methylation of RASSF1A in plasma for the HCC and LC groups showed a significant difference.The AUC of RASSF1A methylation for discriminating the HCC and LC groups was 0.70(0.60-0.79).The AUCs of T1,T2,T3 and T4 HCC and LC were 0.80(0.61,1.00),0.74(0.59-0.88),0.60(0.42-0.79),and 0.68(0.53-0.82),respectively.CONCLUSION RASSF1A methylation in plasma detected by digital PCR may be a potential biomarker for CRC and HCC.

Identification of Shiga-like toxin Escherichia coli isolated from children with diarrhea by polymerase chain reaction

Objective To evaluate the etiology of Shiga-like toxin-producing Escherichia coli (SLTEC) in children with diarrhea. Methods We designed and synthesized 3 pairs of primers located in the SLT1, SLT2, and eaeA genes of enterohaemorrhagic Escherichia coli (EHEC), while the virulent genes SLT1, SLT2, and eaeA from E.coli species were amplified by polymerase chain reaction (PCR). Results One strain of EHEC with SLT1, SLT2, and eaeA in 29 reference strains of diarrhea-causing E.coli (DCEC) and 10 strains of other enterobacteria detected by PCR had positive reactions, while all other DCEC and enterobacteria were negative. Of 474 strains of E. coli isolated from 1032 children with diarrhea and detected by PCR, 20 strains of SLT1 producing E. coli (4.2%) positive, and 7 strains of SLT2 producing E.coli (1.5%) positive; while of 74 strains of entero-SLTs-producing and invasive Escherichia coli (ESIEC), 15 strains of SLT1 (20.3%) and 5 strains of SLT2 (6.8%) were positive. Conclusion Shiga-like toxin E. coli has been identified as a major etiologic agent of children with diarrhea in Taiyuan, China.

序列特异引物引导的聚合酶链式反应(PCR-SSP)结合血清学在疑难血型鉴定中的应用

目的 探讨血型血清学与序列特异引物引导的聚合酶链式反应(PCR-SSP)基因分型在ABO疑难血型鉴定中的作用。方法 用血清学分析鉴定的ABO疑难血型标本80例,同时采用PCR-SSP法进行基因分型,结合两种方法学结果,确定血型及制定输血策略。结果 血清学鉴定亚型40例,其他原因引起的抗原抗体缺失或减弱正常血型40例;PCR-SSP法基因分型亚型41例(3例二者结果不一致:血清学Ael型1例,基因分型O2O2;血清学O型1例,基因分型BO1;血清学A型1例,基因分型AB),正常血型39例。结论 使用血清学结合基因分型鉴定ABO疑难血型具有重要意义。

猪萨佩罗病毒聚合酶链式反应检测方法的建立及应用

猪萨佩罗病毒(Porcine sapelovirus,PSV)是引发猪腹泻的重要病毒之一。为开发一种能快速、准确检测PSV的聚合酶链式反应检测方法,针对PSV的保守区3D基因设计了一对特异性引物,优化了聚合酶链式反应退火温度,分析聚合酶链式反应检测方法特异性和灵敏度,并应用于猪场样品检测。结果表明:建立的聚合酶链式反应检测方法可特异性扩增出PSV目的片段,对其他非靶标猪腹泻病毒则无条带产生,检测低限为5拷贝/μL;在浙江地区采集的88个健康猪粪便样本中检测PSV阳性率为7.95%。该PSV聚合酶链式反应检测方法特异性强、灵敏度高,可应用于PSV前期诊断和防控。

Micro-droplet Digital Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction Technologies Provide Highly Sensitive and Accurate Detection of Zika Virus

The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction(ddPCR) and real-time quantitative PCR(RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold(Ct) value was linear from 10~1 to 10~8 copy/l L, with a standard curve R^2 of 0.999 and amplification efficiency of 92.203%;however, a concentration as low as 1 copy/l L could not be detected. In comparison with RT-qPCR, the dd PCR method resulted in a linear range of 10~1–10~4 copy/l L and was able to detect concentrations as low as 1 copy/l L. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples(above 10~1 copy/l L),while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.

广西道地药材肉桂及阴香的DNA分子鉴定研究

建立一种能够准确鉴别肉桂及阴香的特异性聚合酶链式反应方法,以保障肉桂用药的安全性和有效性。通过对比分析肉桂及阴香的psbA-trnH序列差异找到特异性单核苷酸多态性位点,设计特异性引物,通过优化退火温度、循环次数,评估不同聚合酶种类和不同基因扩增仪等扩增条件对不同来源的肉桂及阴香进行特异性扩增,根据特异性扩增条带进行鉴别。结果表明退火温度为54℃,循环次数为35次时,肉桂经肉桂特异性引物扩增后在100~200 bp处出现特异性条带,阴香无条带;退火温度为56℃,循环次数为40次时,阴香经阴香引物扩增后在200~300 bp处出现特异性条带,肉桂无条带。该研究所建立的特异性PCR方法可以快速准确鉴别出肉桂及阴香,为控制肉桂的质量安全提供参考。

类风湿性关节炎患者外周血单个核细胞IL-10基因启动子甲基化状态研究

白细胞介素-10(interleukin-10,IL-10)是在类风湿性关节炎中发挥重要免疫调节作用的细胞因子,其基因失活与已分化的Th1和Th2细胞染色质结构重塑有关。为了探讨IL-10基因启动子甲基化及基因失活在类风湿性关节炎(Rheumatoid Arthritis,RA)发病和进展中的作用,采用逆转录聚合酶链反应(RT-PCR)、酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)及甲基化特异性聚合酶链反应(MSP),分别检测34例类风湿性关节炎患者和30例健康人外周血单个核细胞IL-10mRNA、蛋白表达水平及基因启动子甲基化状态。结果显示,病例组IL-10mRNA及蛋白表达均低于健康对照组,无统计学差异(P>0.05)。病例组甲基化率(85.29%)高于健康对照组(43.33%),具有统计学差异(χ2=12.439,P=0.000)。IL-10基因启动子甲基化状态与其mRNA表达呈显著负相关(r=?0.579,P=0.001),与所累关节数显著相关,但与血沉(ESR)、C反应蛋白(CRP)、类风湿因子(RF)、年龄均无相关性(P>0.05)。IL-10mRNA表达与年龄、所累关节数、ESR、CRP及RF均无相关性(P>0.05)。上述结果提示,启动子甲基化可能是IL-10基因失活的重要机制,IL-10基因异常高甲基化状态可能参与了RA的发生发展。