Expression of cellular fibronectin mRNA in adult periodontitis and peri-implantitis: a real-time polymerase chain reaction study

Cellular fibronectin （cFn） is a type of bioactive non-collagen glycoprotein regarded as the main substance used to maintain periodontal attachment. The content of cFn in some specific sites can reflect the progress of periodontitis or peri-implantitis. This study aims to evaluate the expression of cFn messenger RNA （mRNA） in tissues of adult periodontitis and peri-implantitis by real-time fluorescent quantitative polymerase chain reaction （PCR） and to determine its clinical significance. A total of 30 patients were divided into three groups of 10： healthy, adult periodontitis and peri-implantitis. Periodontal tissue biopsies （1 mmx I mmx I mm） from each patient were frozen in liquid nitrogen. Total RNA was extracted from these tissues, and the content, purity and integrity were detected. Specific primers were designed according to the sequence, and the mRNA expression levels of cellular fibronectin were detected by real-time PCR. The purity and integrity of the extracted total RNA were both high, and the specificity of amplified genes was very high with no other pollution. The mRNA expression of cFn in the adult periodontitis group （1.526＋0.441） was lower than that in the healthy group （3.253＋0.736）. However, the mRNA expression of cFn in the peri-implantitis group （3.965＋0.537） was significantly higher than that in the healthy group. The difference revealed that although both processes were destructive inflammatory reactions in the periodontium, the pathomechanisms were different and the variation started from the transcription level of the cFn gene.

Real-time polymerase chain reaction for the diagnosis of necrotizing herpes stromal keratitis

AIM： To design, optimize and validate a rapid,internally controlled real-time polymerase chain reaction（RT-PCR） test for herpes simplex virus（HSV） in the diagnosis of necrotizing herpes stromal keratitis.· METHODS： Tears alone or together with corneal epithelium scrapings from 30 patients（30 eyes）suspected of necrotizing herpes stromal keratitis were tested for HSV DNA by RT-PCR. The samples were collected during the first visit and then on the subsequent 7, 14, 28, 42, and 56 d. The symptoms of the patients were scored before treatment to determine the correlation between HSV concentration in the corneal epithelium scrapings and clinical scores.·RESULTS： The positive rate（46.4%） in the corneal epithelium group before the therapy was significantly higher than that（13.3%） in the tears group（P =0.006）.There were 13 positive HSV patients before the therapy,the concentration of HSV DNA in corneal epithelium scrapings group was significantly higher than that in the tears group（paired t-test, P =0.0397）. Multilevel mixedeffects model analysis showed that the difference between the corneal epithelium scrapings group and the tears group was statistically significant（P =0.0049）. The Spearman rank correlation analysis indicated a positive correlation between the HSV concentration in the corneal epithelium scrapings and clinical scores before the treatment（r =0.844, P〈 0.0001）.· CONCLUSION： RT-PCR appears to be a powerful molecular tool for the diagnosis of necrotizing herpes stromal keratitis.

Rapid genotyping of human rotavirus using SYBR green real-time reverse transcription-polymerase chain reaction with melting curve analysis

AIM: To develop a real-time reverse transcriptionpolymerase chain reaction(RT-PCR) assay to genotype rotavirus(G and P) in Alberta from January 2012 to June 2013. METHODS: We developed and validated a different approach to perform rotavirus G and P genotyping using a two-step SYBR green RT-PCR(rt-g PCR) by selecting genotype-specific primers of published conventional RT nested PCR(cn RT-PCR) assay and optimizing the amplification conditions. c DNA was first synthesized from total RNA with Super Script? Ⅱ reverse transcriptase kit followed by amplication step using monoplex SYBR green real-time PCR. After the PCR reaction, melting curve analysis was used to determine specific genotype. Sixteen samples previously genotyped using cn RT-PCR were tested using the new assay and the genotyping results were compared as sensitivity analysis. Assay specificity was evaluated by testing other gastroenteritis viruses with the new assay. The amplicon size of each available genotype was determined by gelelectrophoresis and DNA sequences were obtained using Sanger-sequencing method. After validation and optimization, the new assay was used to genotype 122 pediatric clinical stool samples previously tested positive for rotavirus using electron microscopy between January2012 and June 2013.RESULTS: The new rt-g PCR assay was validated and optimized. The assay detected G1 to G4, G9, G12 and P[4] and P[8] that were available as positive controls in our laboratory. A single and clear peak of melting curve was generated for each of specific G and P genotypes with a Tm ranging from 80 ℃ to 82 ℃. The sensitivity of rt-g PCR was comparable to cn RT-PCR with 100% correlation of the 16 samples with known G and P genotypes. No cross reaction was found with other gastroenteritis viruses. Using the new rt-g PCR assay, genotypes were obtained for 121 of the 122 pediatric clinical samples tested positive for rotavirus: G1P[8](42.6%), G2P[4](4.9%), G3P[8](10.7%), G9P[8](10.7%), G9P[4](6.6%), G12P[8](23.0%), and unknown GP[8](0.8%). For the first time, G12 rotavirus strains were found in Alberta and G12 was the second most common genotype during the study period. Gel electrophoresis of all the genotypes showed expected amplicon size for each genotype. The sequence data of the two G12 samples along with other genotypes were blasted in NCBI BLAST or analyzed with Rota C Genotyping tool(http://rotac.regatools.be/). All genotyping results were confirmed to be correct.CONCLUSION: rt-g PCR is a useful tool for the genotyping and characterization of rotavirus. Monitoring of rotavirus genotypes is important for the identification of emerging strains and ongoing evaluation of rotavirus vaccination programs.

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats

AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

Real-time Fluorescence PCR Method for Detection of Burkholderia glumae from Rice

Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It＇s quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×10^4 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1：100.

Use of real-time polymerase chain reaction for the diagnosis of Pneumocystis pneumonia in immunocompromised patients： a meta-analysis

Background The diagnosis of Pneumocystis pneumonia （PCP） in immunocompromised patients is still challenging today due to the absence of an in vitro culture system and the low diagnostic accuracy of microscopic examinations. Herein, we performed a meta-analysis to evaluate the accuracy of real-time polymerase chain reaction （PCR） in the diagnosis of PCP. Methods We searched Web of Knowledge and Medline from 1990 to May 2010 for studies reporting diagnostic accuracy data regarding the use of real-time PCR in the diagnosis of PCP in immunocompromised patients. Results Ten individual studies were included. Overall, the sensitivity of real-time PCR was 97% （95% CI： 93%-99%）; the specificity was 94% （95% CI： 90%-96%）. The area under the HSROC curve （95% CO for real-time PCR was 0.99 （0.97-0.99）. In a subgroup analysis regarding studies involving HIV patients among the study population, the sensitivity and specificity were 97% （95% CI： 93%-99%） and 93% （95% CI： 89%-96%）, respectively. Regarding studies using Bronchoalveolar lavage （BAL） samples only： sensitivity =98% （95% CI： 94%-99%）; specificity =93% （95% CI： 89%- 96%）, respectively. Regarding studies using microscopy as a reference standard： sensitivity =97% （95% CI： 92%-99%）; specificity =93% （95% CI： 88%-96%）. However, high between-study statistical heterogeneity was observed in all analyses. Conclusions Real-time PCR has a good diagnostic accuracy and may provide a useful adjunctive tool for the diagnosis of PCP in immunocompromised patients. Further studies are needed in order to identify any differences in the diagnostic performance of real-time PCR in HIV and non-HIV immunocompromised patients.

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.

SMRT sequencing and ddPCR reveal the complexity of developmental trajectories and temporal dynamics of gut bifidobacterial communities in infants

Infant intestinal microbiome is closely linked with health and risk of disease. Bifidobacterium are important components of the infant gut and are known to confer various health effects on the host. However, few studies have described the precise composition and dynamics of early infant gut bifidobacterial communities. Thus, this was a pilot study aiming to describe the developmental trajectories and temporal dynamics of bifidobacterial communities in infants before 6 months of age. A total of 28 fecal samples from 4 infants(GF, ZZ, QM, TN, respectively)were collected and analyzed after 5, 15, 30, 60, 90, 120, 150, and 180 days of birth by a bifidobacteria-target method(based on single-molecule real-time sequencing of partial bifidobacterial rpsK genes)in conjunction with droplet digital polymerase chain reaction(ddPCR). The infant fecal microbiota comprised a total of 11 bifidobacterial species, including 4 major species, i.e., B. dentium(37.35%), B. catenulatum(32.04%), B. breve(22.24%), and B. animalis(8.02%). The infant microbiota showed highly individualized developmental trajectories. The leading species for GF was B. catenulatum, with a relatively stable developmental trajectory. In ZZ, B. breve was enriched, and the developmental trajectory was rather fluctuating. The most abundant species for QM and TN was B. dentium. The developmental trajectory of B. dentium in QM showed a trend of gradual decrease, whereas an opposite trend was seen in samples of TN. The results of ddPCR confirmed large variations in quantities of bifidobacteria between infants and suggested discordances in temporal dynamics of bifidobacterial communities during the first half year of infancy. In conclusion, our results suggested that the early infant gut bifidobacterial microbiota was highly complex and temporal dynamics, with individualized developmental trajectories, which should be considered in future research of infant gut microbiota.

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。

转基因玉米59122品系的特异性检测

使用反向聚合酶链式反应(PCR)技术克隆了转基因玉米59122的外源基因与玉米基因组之间的两段侧翼序列,并据其左侧侧翼序列设计了具品系特异性的引物,运用半巢式PCR技术建立了59122的品系特异性二重PCR检测方法,扩增片段100bp,横跨pat终止子与转基因玉米侧翼基因之间。以转基因玉米59122、MON863、MON810、GA21、NK603,转基因大豆Roundup Ready和转基因油菜GT73等为材料,证明本方法与其他转基因作物具有高特异性。本方法在检测59122时,确定出连接体系中线性DNA的最佳质量浓度为1ng/μL左右,检出限达到0.1%,灵敏度为38个单倍体基因组拷贝数。因此可准确、快速、高效地检测转基因玉米及其产品,或作为常规PCR定性检测后的验证方法。

复合式蝎形引物实时定量检测端粒酶延伸产物

针对端粒酶延伸产物中靶基因序列的特殊性 ,开发了一种可产生荧光的复合式蝎形引物 ,该引物的 5′端带有可特异性检测靶基因的探针序列 ,PCR阻断剂将其与引物序列连接 .当复合式蝎形引物延伸 ,探针序列与同一分子内的靶基因杂交 ,荧光信号产生 .运用该技术 ,建立了定量检测端粒酶延伸产物的实时荧光PCR方法 .该法可在快速PCR循环条件下 ,对 0 .15～ 1.5 0× 10 3 amol/ μL范围内的样品进行定量检测 ,线性相关系数R2 =0 .9992 .该法操作简便 。

基于双重PCR技术的鹿茸及其伪品DNA指纹特征和鉴定

目的:分析鹿茸线粒体细胞色素b(Cytb)和细胞色素C氧化酶亚基Ⅰ(COⅠ)基因特异性,建立双重PCR技术鉴别鹿茸真伪的分子指纹特征。方法:利用碱变性法提取梅花鹿茸、马鹿茸、驯鹿茸和新西兰鹿茸的基因组DNA,应用引物设计软件Premier 5.0针对Cytb和COⅠ分别设计特异性引物(分别为Cytb 1、2和COⅠ1、2、3),采用单一及双重引物分别进行PCR扩增,筛选特异性强的引物,确定最佳PCR反应条件。结果:采用碱变性法提取的鹿茸基因组DNA片段长度为23 000bp,DNA纯度即A(260)/A(280)为1.80±0.02;应用单一引物进行PCR扩增无法鉴定鹿茸的真伪,而引物Cytb 1和COⅠ1组合后,解链温度为58℃时,梅花鹿茸(吉林、安徽)、马鹿茸均能扩增出395和525bp大小的2个片段,而驯鹿茸和新西兰鹿茸均未能扩增出相应片段;采用该提取方法及最优化的PCR反应条件,对市售样品进行检测,检测结果与实际情况完全一致。结论:双重PCR技术可从分子水平鉴别鹿茸的真伪,该方法特异性高、实用性强,且简便快捷,在鹿茸真伪鉴别方面具有较高的应用价值。

副溶血弧菌和溶藻弧菌双重PCR检测方法的建立与初步应用

为建立同时快速检测海产品中的副溶血弧菌(VP)和溶藻弧菌(VA)的双重PCR方法,本研究根据VP和VA的toxR基因序列设计针对这两种细菌的两对特异性引物,建立能够快速同时检测这两种细菌的双重PCR方法,并对该反应体系的特异性和灵敏度进行检测。结果显示纯培养细菌VP和VA的检测灵敏度分别为2.32×103cfu/mL和2.56×103cfu/mL,临床病料检测灵敏度分别为2 cfu和3 cfu;与枸橼酸杆菌、沙门氏菌、美人鱼弧菌、霍乱弧菌、麦氏弧菌无交叉反应。研究表明本实验方法操作简便快速、特异性强、灵敏度高、稳定性好,并且经济实惠,值得推广应用。

嗜肺军团菌与非嗜肺军团菌所致社区获得性肺炎的临床对比分析

目的探讨嗜肺军团菌与非嗜肺军团菌感染所致社区获得性肺炎的临床特点,提高对军团菌肺炎的认识。方法采用双重聚合酶链反应(DPCR)法对根据临床表现及血清抗体结果,临床诊断为军团菌肺炎患者的痰标本进行军团菌DNA检测。选择痰DPCR和血清抗体结果呈一致阳性的患者并根据检测结果将其分为嗜肺军团菌肺炎组(42例)和非嗜肺军团菌肺炎组(18例)。将2组患者的临床资料进行对比分析。结果痰标本DPCR结果与血清特异性抗体结果呈一致阳性,嗜肺军团菌肺炎与非嗜肺军团菌肺炎诊断明确。嗜肺军团菌肺炎多发于既往体健的青壮年,主要发病于夏秋季;非嗜肺军团菌倾向于四季散发,多发于有基础疾病的老年人。与非嗜肺军团菌肺炎相比,嗜肺军团菌肺炎患者的肺脏受累面积较大,胸腔积液及ARDS发病率高,同时全身感染中毒症状和肺外表现更为明显(P<0.05)。结论嗜肺与非嗜肺军团菌感染所致社区获得性肺炎临床特点有所不同,早期予大环内酯类/喹诺酮类抗生素治疗是改善预后的关键手段。

马铃薯、番茄内源基因PCR检测引物设计及特异性分析

对马铃薯UDP-葡萄糖焦磷酸化酶基因(UGPase,U20345)和番茄多聚半乳糖醛酸酶-2a基因(PG-2a,X04583.1)进行序列相似性分析和多序列比对,确定特异片段,设计特异引物,经聚合酶链式反应(polymerase chain reaction,PCR)检测,引物具有较高的特异性;同时建立利用双重PCR检测马铃薯和番茄混合成分的方法,当马铃薯和番茄DNA混合质量为1.25ng时,仍可对其成分进行可靠的鉴定。新设计的马铃薯和番茄内源基因的引物及建立的双重PCR检测方法具有一定的特异性和灵敏性,适于出入境检验检疫部门对马铃薯和番茄制品进行检测。

人巨细胞病毒的PCR检测

①目的 寻找早期快速检测人类巨细胞病毒 (HCMV)感染的敏感方法。②方法 用HCMV即刻早期 (IE)基因和晚期 (LA)基因的 7对特异性引物分别进行普通聚合酶链反应 (PCR)、套式PCR(nPCR)和双重PCR ,检测血清中的HCMVDNA ,比较不同引物及不同PCR方法的灵敏度。③结果 同一基因的不同引物扩增HCMVDNA灵敏度差别无显著意义 ;LA基因的引物扩增灵敏度高于IE基因的引物 ;3种方法中以nPCR灵敏度最高 ,其次为双重PCR ,普通PCR最低。④结论 用LA基因的特异性引物进行nPCR是早期快速检测HCMV感染的敏感方法。

MecA、femB基因PCR联合扩增法检测耐甲氧西林金黄色葡萄球菌

目的了解mecA、femB多重聚合酶链反应(PCR)法对耐甲氧西林金黄色葡萄球菌(MRSA)的检出效率。方法对临床分离的71株非重复的金黄色葡萄球菌,采用多重PCR进行检测。结果 mecA、femB多重聚合酶链反应对MRSA的检测的特异性为100%,敏感性为97.87%。结论 mecA、femB多重PCR法可以对临床分离的金黄色葡萄球菌的甲氧西林耐药作出判断,是一种高效、敏感、特异性高的检测技术。

结核分枝杆菌和牛分枝杆菌的二重PCR快速检测试剂盒的研究

根据已发表的结核分枝杆菌23S rRNA和牛分枝杆菌特殊基因序列,设计并合成了2对可扩增结核分枝杆菌和牛分枝杆菌特异性基因片段的引物,建立二重PCR快速检测结核分枝杆菌和牛分枝杆菌的方法,并研制出检测试剂盒。结果表明,该检测试剂盒对结核分枝杆菌能特异性地扩增出838 bp条带而扩增不出631 bp条带,牛分枝杆菌能特异地扩增出838 bp和631 bp条带,而对其他牛菌株的DNA扩增结果均为阴性;敏感性试验表明最低能检测出50 pg的结核分枝杆菌和牛分枝杆菌DNA模板;保存期测定结果表明,在-20℃条件下保存至1、3、6、9个月时,试剂盒的敏感性无明显变化,仍能检测到50 pg的DNA模板,保存至12个月时其敏感度仍能100%检出阳性样品。

新生儿谷胱苷肽S转移酶μ1基因缺失状况的研究

谷胱苷肽S转移酶μ1（glutathioneStransferasesμ1，CSTμ1）对解毒香烟烟雾中的苯并（a）芘等致癌物有重要作用。现已证实正常人中约有50％缺失这种基因，这暗示将增加了肺癌发病的危险性。为了达到早期预防与预测，给新生儿以优育指导。本文采用双重PCR技术对106例新生儿脐血DNA进抒GSTμ1基因检测，并以健康人为对比，研究其缺失率为44．7％，同时新生儿与健康成人不同年龄组、新生儿男、女性别与成人男、女性别缺失率比较，经统计学卡方检验均无显著差异。提示GSTμ1基因缺失是新生儿自身遗传性不良因素，他（她）们如生活在香烟烟雾环境中，发生肿瘤危险性比无GSTμ1缺失的新生儿可能性大。并且通过本研究建立一种快速、准确的基因检测方法，有利于广泛群体筛查，应用临床及科学地指导优育有一定重要意义。

一种检测KPC和NDM耐药基因的双重PCR-LF方法建立及初步应用

目的建立一种双重聚合酶链反应-侧流层析(PCR-LF)方法快速检测肺炎克雷伯菌碳青霉烯酶(KPC)和新德里金属β-内酰胺酶(NDM)两种耐药基因。方法根据KPC和NDM的基因保守区,分别设计并合成了特异性引物,建立双重PCR和LF技术相结合的检测体系,通过优化扩增和LF检测体系,将建立起的方法初步应用于临床标本检测。结果双重PCR-LF可用于KPC和NDM两种碳青霉烯酶基因的检测,检测下限均为200 CFU/mL。以药敏表型检测为参考方法,利用双重PCR-LF直接检测120份临床无菌体液标本,两种方法结果差异无统计学意义(P>0.05),Kappa值为0.644,表明一致性较好。结论双重PCR-LF方法用于KPC和NDM两种耐药基因检测快速有效,对于指导临床用药与监控院内细菌碳青霉烯类抗菌药物耐药性的传播具有重要意义。