Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay

A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic H1N1/2009 virus infection.

A quantitative polymerase chain reaction assay for the enumeration of brown tide algae Aureococcus anophagefferens in coastal waters of Qinhuangdao

Aureococcus anophagefferens, a small pelagophyte algae, has caused brown tide blooms in coastal waters of Qinhuangdao in recent years, presenting significant negative impacts on the shellfish mariculture industry. Under standard light microscopy, it is visually indistinguishable from other small algae in field samples due to its extremely small size. In this study, quantitative polymerase chain reaction（q PCR） based on 18 S r DNA sequences was developed and used to detect and enumerate A. anophagefferens. A linear regression（R2 = 0.91） was generated based on cycle thresholds value（Ct） versus known concentrations of A. anophagefferens. Twenty-two field samples collected in coastal waters of Qinhuangdao were subjected to DNA extraction and then analyzed using q PCR. Results showed that A. anophagefferens had a wide distribution in coastal waters along Qinhuangdao. Elevated A. anophagefferens abundance, category 3 brown tide blooms（〉200 000 cells/m L） occurred at Dongshan Beach and Tiger-stone Beach in August in 2013. In shellfish mariculture areas along coastal waters of Qinhuangdao, 4 stations had category 3 blooms, and 6 stations had category 2 blooms（35 000–200 000 cells/m L） in August and all stations had category 1 blooms（〉0 to ≤35 000 cells/m L） in October. Quantitative PCR allows for detection of A. anophagefferens cells at low levels in filed samples, which is essential to effective management and prediction of brown tide blooms.

RT-qPCR normalization of reference genes in different lifehistory stages of Gracilaria vermiculophylla(Rhodophyta)

The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.

Reference genes for quantitative RT-PCR data in gastric tissues and cell lines

AIM:To evaluate the suitability of reference genes in gastric tissue samples and cell lines.METHODS:The suitability of genes ACTB,B2M,GAPDH,RPL29,and 18S rRNA was assessed in21 matched pairs of neoplastic and adjacent nonneoplastic gastric tissues from patients with gastric adenocarcinoma,27 normal gastric tissues from patients without cancer,and 4 cell lines using reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR).The ranking of the best single and combination of reference genes was determined by NormFinder,geNorm,BestKeeper,and DataAssist.In addition,GenEx software was used to determine the optimal number of reference genes.To validate the results,the mRNA expression of a target gene,DNMT1,was quantified using the different reference gene combinations suggested by the various software packages for normalization.RESULTS:ACTB was the best reference gene for all gastric tissues,cell lines and all gastric tissues plus cell lines.GAPDH+B2M or ACTB+B2M was the best combination of reference genes for all the gastric tissues.On the other hand,ACTB+B2M was the best combination for all the cell lines tested and was also the best combination for analyses involving all the gastric tissues plus cell lines.According to the GenEx software,2 or 3 genes were the optimal number of references genes for all the gastric tissues.The relative quantification of DNMT1 showed similar patterns when normalized by each combination of reference genes.The level of expression of DNMT1 in neoplastic,adjacent non-neoplastic and normal gastric tissues did not differ when these samples were normalized using GAPDH+B2M(P=0.32),ACTB+B2M(P=0.61),or GAPDH+B2M+ACTB(P=0.44).CONCLUSION:GAPDH+B2M or ACTB+B2M is the best combination of reference gene for all the gastric tissues,and ACTB+B2M is the best combination for the cell lines tested.

Establishment of a new quantitative detection approach to adefovir-resistant HBV and its clinical application

AIM:To establish the more feasible and sensitive assessment approach to the detection of adefovir (ADV) resistance-associated hepatitis B virus (HBV) quasispecies.METHODS: Based on the characteristics of rtA181V/T and rtN236T mutations, a new approach based on real-time fluorescent quantitative polymerase chain reaction (RT-PCR) was established for the detection of ADV-resistant HBV quasispecies, total HBV DNA, rtA181 and rtN236 mutations in blood samples from 32 chronic hepatitis B (CHB) patients with unsatisfactory curative effect on ADV and compared with routine HBV DNA sequencing.RESULTS: Both the sensitivity and specificity of this new detection approach to ADV-resistant HBV quasispecies were 100%, which were much higher than those of direct HBV DNA sequencing. The approach was able to detect 0.1% of mutated strains in a total plasmid population. Among the 32 clinical patients, single rtA181 and rtN236T mutation and double rtA181T and rtN236T mutations were detected in 20 and 8, respectively, while ADV-resistant mutations in 6 (including, rtA181V/T mutation alone in 5 patients) and no associated mutations in 26.CONCLUSION: This new approach is more feasible and efficient to detect ADV-resistant mutants of HBV and ADV-resistant mutations before and during ADV treatment with a specificity of 100% and a sensitivity of 100%.

Quantitative Analysis of ATP Sulfurylase and Selenocysteine Methyltransferase Gene Expression in Different Organs of Tea Plant (<i>Camellia sinensis</i>)

Tea plant (Camellia sinensis) has unique biological features for the study of cellular and molecular mechanisms, an evergreen broad-leaved woody plant which can accumulate selenium in soil abundant of Selenium. Expression of the genes related to Selenium (Se) metabolism is an adaptation to the soil environment for a long period. The purpose of the present study was to explore if there exist differences of expression about these genes in tea plant between growing in Selenium-abundant and normal soil. A quantitative real-time reverse transcription polymerase chain reaction (Q-RT-PCR) assay was done for quantification of ATP sulfurylase (APS) and selenocysteine methyltransferase (SMT) mRNA normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in tea plant. Young leaves, mature leaves and tender roots from tea plants growing in soil abundant of Selenium were respectively obtained from Shitai County, Anhui Province, and also the relevant materials of the selenium un-enriched tea plant planted at agricultural garden of Ahui Agriculture University were taken as control for real-time PCR analysis. The results showed that APS1, APS2 and SMT expression levels for either young or mature leaves in selenium-enriched tea plant were lower than that in ordinary (selenium un-enriched) tea plant. In contrast, the APS1, APS2 and SMT expression level of roots in selenium-enriched tea plant were all higher than that in ordinary tea plant. APS1 gene expression level of roots in selenium-enriched tea plant was about 1.6 times higher than that in the ordinary tea plant, APS2 gene expression level was about 4.8-fold higher than that in the ordinary tea plant, SMT gene expression level was about 3.3 times higher than that in the ordinary tea plant. Among various tissues of selenium-enriched tea plant, APS1 gene relative expression level of young leaves was similar to or slightly higher than mature leaves, and the one of roots was the lowest among them;APS2 gene relative expression level of young leaves was similar to or slightly higher than the roots, and the one of mature leaves was the lowest among them;SMT gene relative expression level of young leaves was similar to or slightly higher than mature leaves, and the one of roots was the highest among them. Our results suggest that there existed correlation between selenium and expression levels of these genes.

Quantitative analysis of a panel of gene expression in prostate cancer——with emphasis on NPY expression analysis

Objective: To investigate molecular alterations associating with prostate carcinoma progression and potentially provide information toward more accurate prognosis/diagnosis. Methods: A set of laser captured microdissected (LCM) speci- mens from 300 prostate cancer (PCa) patients undergoing radical prostatectomy (RP) were defined. Ten patients representing "aggressive" PCa, and 10 representing "non-aggressive" PCa were selected based on prostate-specific antigen (PSA) recurrence, Gleason score, pathological stage and tumor cell differentiation, with matched patient age and race between the two groups. Normal and neoplastic prostate epithelial cells were collected with LCM from frozen tissue slides obtained from the RP specimens. The expressions of a panel of genes, including NPY, PTEN, AR, AMACR, DD3, and GSTP1, were measured by quantitative real-time RT-PCR (TaqMan), and correlation was analyzed with clinicopathological features. Results: The expressions of AMACR and DD3 were consistently up-regulated in cancer cells compared to benign prostate epithelial cells in all PCa patients, whereas GSTP1 expression was down regulated in each patient. NPY, PTEN and AR exhibited a striking difference in their expression patterns between aggressive and non-aggressive PCas (P=0.0203, 0.0284, and 0.0378, respectively, Wilcoxon rank sum test). The lower expression of NPY showed association with "aggressive" PCas based on a larger PCa patient cohort analysis (P=0.0037, univariate generalized linear model (GLM) analysis). Conclusion: Despite widely noted heterogeneous nature of PCa, gene ex- pression alterations of AMACR, DD3, and GSTP1 in LCM-derived PCa epithelial cells suggest for common underlying mecha- nisms in the initiation of PCa. Lower NPY expression level is significantly associated with more aggressive clinical behavior of PCa; PTEN and AR may have potential in defining PCa with aggressive clinical behavior. Studies along these lines have potential to define PCa-associated gene expression alterations and likely co-regulation of genes/pathways critical in the biology of PCa onset/progression.

扩展青霉菌实时定量PCR内参基因挖掘与应用

实时定量聚合酶链式反应(polymerase chain reaction,PCR)技术因其高效且便捷的特点在基因表达检测中被广泛应用。实时定量PCR结果数据处理策略之一是使用内参基因进行基因表达数据的标准化。文章基于扩展青霉菌孢子不同生长阶段的RNA-seq数据,挖掘和注释了694个稳定表达的候选内参基因;随机挑选出Knr4、Isy1、Spt5、Nucb、Hp1、Hp2、Whth、Hp3、Gph3、Pwi、Taf4、Hp4、Asy、GTPase在内的14个基因,以4、25℃条件下PDB培养基生长6、12、24、36 h的扩展青霉菌为材料,进行实时定量PCR实验。实时定量PCR数据基于geNorm和NormFinder 2种软件的综合分析,表明Isy1、Spt5、Nucb、Hp1适合作为内参基因。以Isy1和Spt5分别作为内参基因,检测Gh30基因在4、25℃条件下扩展青霉菌生长发育过程中的基因表达,显示出一致的基因表达水平,进一步验证了挖掘得到的内参基因的可靠性。该研究为扩展青霉菌的分子生物学研究提供了优良的内参基因,同时提供了一种高效的内参基因的挖掘和鉴定方法。

Sequence Analysis and Quantitative Detection of Norwalk-like Viruses in Cultured Oysters of China

We isolated 4 Norwalk-like viruses (NLVs) contaminated oysters from 33 Chinese oysters collected from local commer- cial sources of Shandong Province. After amplification of the RNA-dependent RNA polymerase (RdRp) region of NLVs genomes with RT-PCR, the open reading frame 1 (ORF1) of the RdRp was sequenced and subjected to multiple-sequence alignment. The re- sults showed that NLVs in the four isolates belong to genogroup II. The sequence comparison showed that the similarity between four Chinese oyster isolates were higher than 99.0%, which indicated that NLVs prevalent in close areas have high homogeneity in genome sequences. In addition, the most conserved sequences between diverse NLVs were used to design primers and TaqMan probes, then the real-time quantitative PCR assay was performed. According to the standard curve of GII NLVs, the original amounts (copies) of NLVs in positive patient’s fecal isolate, positive Japanese oyster isolate, and the Chinese oyster isolate were 8.9×108, 1.25×108 and 4.7×101 respectively. The detecting limit of NLVs was 1×101 copies. This study will be helpful for routine diagnosis of NLVs pathogens in foods and thus for avoiding food poisoning in the future.

Design and use of group-specific primers and probes for real-time quantitative PCR

Real-time quantitative polymerase chain reaction(qPCR)has gained popularity as a technique to detect and quantify a specific group of target microorganisms from various environmental samples including soil,water,sediments,and sludge.Although qPCR is a very useful technique for nucleic acid quantification,accurately quantifying the target microbial group strongly depends on the quality of the primer and probe used.Many aspects of conducting qPCR assays have become increasingly routine and automated;however,one of the most important aspects,designing and selecting primer and probe sets,is often a somewhat arcane process.In many cases,failed or non-specific amplification can be attributed to improperly designed primer-probe sets.This paper is intended to provide guidelines and general principles for designing group-specific primers and probes for qPCR assays.We demonstrate the effectiveness of these guidelines by reviewing the use of qPCR to study anaerobic processes and biologic nutrient removal processes.qPCR assays using group-specific primers and probes designed with this method,have been used to successfully quantify 16S ribosomal Ribonucleic Acid(16S rRNA)gene copy numbers from target methanogenic and ammoniaoxidizing bacteria in various laboratory-and full-scale biologic processes.Researchers with a good command of primer and probe design can use qPCR as a valuable tool to study biodiversity and to develop more efficient control strategies for biologic processes.

实时荧光定量PCR定量方法研究进展

实时荧光定量PCR以其特异性强、灵敏度高、重复性好、定量准确、速度快、全封闭反应等优点而成为了分子生物学研究中的重要工具,综述了实时荧光定量PCR技术及其定量方法的研究进展,并展望了其应用前景。

单增李斯特菌和金黄色葡萄球菌双重荧光定量PCR检测方法建立

根据GenBank公布的单增李斯特菌的hlyA基因序列和金黄色葡萄球菌的nuc基因序列各设计一对引物和一条探针,建立基于TaqMan探针的单增李斯特菌和金黄色葡萄球菌双重荧光定量聚合酶链式反应(PCR)检测方法。该方法对所有目标菌株均产生特异性扩增曲线,其他非目标菌均不产生扩增曲线,具有较强的特异性。且该法对阳性重组质粒pMD18-hlyA和pMD18-nuc同时定量扩增的敏感度分别为19.5拷贝/μL和18.7拷贝/μL。同步检测人工染菌肉样中单增李斯特菌和金黄色葡萄球菌的最低检出限均为102CFU/g。

SYBR荧光实时定量PCR检测非小细胞肺癌组织与外周血中RRM1和ERCC1及BRCA1基因表达水平

目的:建立荧光实时定量PCR技术,检测非小细胞肺癌组织与外周血RRM1和ERCC1及BRCA1基因表达水平。方法:分别构建RRM1、ERCC1和BRCA1及管家基因β-actin质粒标准品,以SYBR荧光实时定量PCR分析,制备标准曲线,对非小细胞肺癌组织与外周血中RRM1、ERCC1和BRCA1及管家基因β-actin的mRNA进行检测。结果:标准曲线呈良好的线性关系。标准品的熔解曲线均呈单峰,特异性良好,说明基本无非特异性扩增。结论:所建SYBR荧光实时定量PCR方法操作简便,费用低,特异性好,准确度、灵敏度高,为后续研究构建了理想的平台。

耐热菌的竞争定量PCR检测方法优化与建立

【目的】研究苹果浓缩汁中耐热菌(Alicyclobacillusacidoterrestris)的定量PCR快速检测法。【方法】通过引物设计、PCR扩增及凝胶纯化试剂盒回收,构建并获得耐热菌的PCR竞争模板;用获得的竞争模板作为定量内标物建立耐热菌的竞争定量PCR(QC-PCR)检测体系。【结果】经对建立的QC-PCR检测体系优化,目标模板检测灵敏度由5×104个分子/PCR体系,提高到50个分子/PCR体系,竞争模板和目标模板分子共扩增可检测到5×102个目标模板分子/PCR体系,并能从人工回添苹果浓缩汁样品中定量检测到5×103cfu/PCR体系的耐热菌,整个检测时间为4～5h,比传统的细菌培养皿培养记数法时间(4～5d)大大缩短。【结论】本研究建立的耐热菌竞争定量PCR检测法特异、快速,可作为微生物PCR竞争模板构建和建立竞争定量PCR的方法学参考,也可用于商品果汁和苹果浓缩汁工业化生产中耐热菌的快速检测和质量安全控制。

实时荧光定量PCR技术监测腌制麻竹笋中乳酸乳球菌动态变化

采用实时荧光定量聚合酶链式反应（quantitative real-time polymerase chain reaction,q RT-PCR）技术定量监测6 g/100 m L盐质量浓度腌制麻竹笋中乳酸乳球菌（Lactococcus lactis）的动态变化。经乳酸乳球菌标准菌株基因组DNA提取、标准阳性质粒制备、标准曲线绘制、各时期竹笋腌制发酵液中细菌基因组DNA提取和乳酸乳球菌qRTPCR特异性扩增,对腌制液中乳酸乳球菌进行定量检测。结果表明,在腌制过程中（0-63 d）,随着腌制时间的延长,乳酸乳球菌含量逐渐升高,在腌制14 d时达到最大值（4.63×10^8 copies/μL）,与0 d（2.41×10^2 copies/μL）相比增加了6个数量级,而后浓度缓慢降低,在腌制63 d时浓度为5.02×10^6 copies/μL。qRT-PCR技术为定量监测腌制麻竹笋中微生物的动态变化提供了一条可靠、快速的有效途径。

产毒微囊藻mcyA基因荧光定量PCR方法的建立

针对微囊藻毒素合成酶基因mcyA探索一种适用于自然水样中微囊藻毒素产毒潜能检测的TaqMan实时荧光定量PCR方法。该方法从铜绿微囊藻FACHB-1279中的mcyA基因中扩增出目的基因片段,用TA克隆方法将基因片段插入Allel TA vector(ABP-CE-TAVEC020)中来制备质粒标准品。通过不同连续稀释倍数的质粒标准品成功构建了mcyA基因的标准曲线,该法构建的标准曲线相关系数高达0.998 3,满足实时荧光定量RT-PCR的要求。该方法检测所需水样少(仅200μL),具有水样基因组提取操作简便、快速、线性范围广、灵敏度高等优点,能应用于水库、湖泊等水源中具有产毒性能的微囊藻的快速定量检测。

实时荧光定量PCR法与常规PCR法及细菌培养法检测单增李斯特菌的比较

目的:比较实时荧光定量PCR法、常规PCR法及细菌培养法检测单核细胞增生性李斯特菌的灵敏度与特异性。方法:采用建立的实时荧光定量PCR、常规PCR及传统细菌培养法3种方法,同时对单核细胞增生性李斯特菌等细菌进行检测。结果:实时荧光定量PCR检测的灵敏度可达19 cfu/ml,且有很高的特异性,对英诺克李斯特菌等10种相关细菌均无交叉反应,从细菌核酸提取至完成检测仅需3 h左右。结论:实时荧光定量PCR检测由于在密封环境中进行,避免了产物与环境间的交叉污染,且是3种方法中最为快速敏感的方法,适用于公共卫生应急疫情的实验室快速诊断。

采用荧光定量PCR溶解曲线法监测人TCR alpha链CDR3谱系漂移技术的初步探讨

目的:初步探讨荧光定量PCR溶解曲线分析技术监测人外周血T细胞TCRalpha链CDR3谱系漂移(单/寡/多克隆增生)。方法:提取4例正常人、2例淋巴瘤型白血病患者PBMC中的总RNA,逆转录成cDNA,以32个人TCRalpha链胚系可变区基因家族(TRAV)设计上游引物,共同的TCRalpha胚系链恒定区基因家族(TRAC)设计下游引物,荧光定量PCR(FQ-PCR)扩增32个TRAV基因各家族CDR3谱系,溶解曲线法分析各家族CDR3谱系的单/寡/多克隆增生。结果:正常人外周血T细胞TCRalpha链32个家族CDR3表达频率不一致,各家族PCR产物的溶解曲线谱型图(melting curve spectratyping)呈现熔点不同的CDR3多态性,为多克隆增生的高斯分布,2例淋巴瘤型白血病患者外周血T细胞TCRalpha链32个家族CDR3表达频率不一致,部分家族呈缺失状态,患者各家族PCR产物的溶解曲线谱型图上,多数家族为多克隆增生的高斯分布,但每个患者均出现数量不等的单克隆和寡克隆增生家族。结论:荧光定量PCR溶解曲线分析TCRalpha链CDR3谱系漂移技术方法稳定简便,能较好地监测正常人和临床样本外周血T细胞TCRalpha链CDR3谱系漂移(单/寡/多克隆增生)。

QF-PCR快速产前诊断常见非整倍体病的价值

目的评价荧光定量聚合酶链反应(OF-PCR)技术在产前快速诊断常见染色体非整倍体病的临床价值。方法选取2013年1月至2014年6月在我院进行产前诊断的孕妇1 035例,所有孕妇均接受染色体核型分析及QF-PCR检查,比较并分析两种检验结果的符合性。结果染色体核型分析正常的孕妇1 008例中有4例QF-PCR怀疑异常,其中3例经增加短串联重复序列(STR)位点再次分析,结果正常,1例随访未见异常。18例21、18、13、X、Y染色体非整倍体两者结果一致(包括1例18三体嵌合体及1例易位型21-三体),符合率为100%。结论 QF-PCR能快速高效的检出染色体非整倍体,但仍有误诊可能,选择适宜的STR组合可以提高检测效率,QF-PCR作为染色体核型分析的有效补充在快速产前诊断中有重要价值。

自身淬灭探针荧光定量PCR检测淋病奈瑟菌

目的利用自身淬灭探针技术建立敏感、特异、快速、价廉且能广泛应用的淋病奈瑟菌荧光定量PCR检测方法。方法构建重组质粒pGEM-11Zf-CPPB作为标准品,自行设计自身淬灭探针,建立、优化定量PCR体系,并进行方法学评价及临床应用。结果所建立方法的线性检测范围为101～109拷贝/μL,灵敏度为10拷贝/μL,特异性为100%。天间变异系数(CV)为2.38%,批内CV为1.32%,批间CV为2.75%。该方法比培养方法更快速、灵敏。结论以自身淬灭探针技术为平台的淋病奈瑟菌荧光定量PCR方法灵敏度高、特异性好,对淋病的早期快速诊断有较高价值。