Quantitative real-time RT-PCR detection for CEA, CK20 and CK19 mRNA in peripheral blood of colorectal cancer patients

This study is aimed at establishing a sensitive approach to detect disseminated tumor cells in peripheral blood and evaluate its clinical significance. A total of 198 blood samples including 168 from colorectal carcinoma (CRC) patients and 30 from healthy volunteers were examined by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to evaluate the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and cytokeratin 19 (CK19) mRNA. CEA mRNA was detected in 35.8% of patients and 3.3% of controls, CK20 mRNA in 28.3% of patients and 6.7% of controls, and CK19 mRNA in 41.9% of patients and 3.3% of controls. CEA and CK20 mRNA positive ratio increased with the advancing Dukes stages, but there was no significant difference in positive ratio between any two stages (P>0.05). Also, relatively high positive ratio of CEA, CK20 and CK19 mRNA expression was observed in some CRC patients with earlier Dukes stages. A higher positive ratio was obtained when two or three detection markers were combined compared to a single marker. Our study indicates that quanti-tative real-time RT-PCR detection for CEA, CK20 and CK19 mRNA in peripheral blood is a valuable tool for monitoring early stage dissemination of CRC cells in blood circulation.

Detection of Survivin mRNA in nasopharyngeal carcinoma by real-time fluorescence quantitative RT-PCR

Objective： To establish the method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in nasopharyngeat carcinoma （NPC） tissues. Methods： The total RNA was extracted from NPC cell line CNE-2 and tissues with Trizol and then been transcribed reversely to cDNA, a method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in NPC tissues had been established, in which chronic nasopharyn-gitis patients＇ nasopharynx tissues treated as control group. Results： The expression of Survivin mRNA all could be detected either in CNE-2 cells, NPC tissues or in chronic nasopharyngitis patients＇ nasopharynx tissues, and there was higher the expression level of Survivin mRNA in NPC tissues than which in chronic nasopharyngitis patients＇ nasopharynx tissues, the difference was significant （P 〈 0.01）. The expression of Survivin mRNA could be detected both in stage Ⅰ ＋ Ⅱ and stage Ⅲ ＋ Ⅳ NPC, and there was no significant difference in relative quantifications of gene expression between these two groups （P 〉 0.05）. There was no relationship between Survivin mRNA expression and age and sex of NPC patients （P 〉 0.05）. Conclusion： Real time fluorescence quantitative RT-PCR is a rapid, effective and high sensitive method for detecting the expression of Survivin mRNA in NPC tissues. The overexpression of Survivin mRNA may play some roles in pathogenesis of NPC.

Detection and clinical significance of multidrug resistance-1 mRNA in bone marrow cells in children with acute lymphoblastic leukemia by real-time fluorescence quantitative RT-PCR

Objective： Multidrug resistance（MDR） is one of the most important reasons for treatment failure and recurrence of acute leukemia. Its manifestations are different in children with acute lymphoblastic leukemia（ALL） which may be due to different detection methods. This study was to detect the expression of MDR1 mRNA in bone marrow cells of children with ALL by real-time fluorescence- quantitative reverse transcription polymerase-chain reaction（FQ-RT-PCR）, and combine minimal residual desease（MRD） detection by flow cytometry（FCM） and to study their relationship with treatment response and prognosis of ALL. Methods：The MDR1 mRNA levels in bone marrow cells from 67 children with ALL[28 had newly diagnosed disease, 27 had achieved complete remission（CR）, 12 recurrent] and 22 children without leukemia were detected by FQ-RT-PCR. MRD was detected by FCM. The patients were observed for 9-101 months, with a median of 64 months. Results：Standard curves of human MDR1 and GAPDH genes were constructed successfully. MDR1 mRNA was detected in all children with a positive rate of 100%. The mRNA level of MDR1 was similar among the newly diagnosed ALL group, CR group, and control group（P 〉 0.05）, but significantly higher in the recurrence group than that in newly diagnosed disease group and control group（0.50 ± 0.55 vs. 0.09 ± 0.26 and 0.12 ± 0.23, P〈 0.05）. 54 ALL patients were followed up, and it was found that MDR1 mRNA level was significantly higher in ALL patients within 3 years duration than that of ALL patients with 3-6 years and over 6 years duration（0.63 ± 0.56 vs. 0.11 ± 0.12 and 0.04 ± 0.06, P〈 0.01）. For the 28 children with newly diagnosed disease, the MDR1 mRNA level was similar between WBC 〉 50 ~ 109 group and WBC〈50 × 10^9 group（P〉 0.05）. In the 33 CR patients, the MDR1 mRNA level was significantly higher in MRD〉10a group than that in MRD〈10a group（0.39 ± 0.47 vs. 0.03 ± 0.03, P 〈 0.05）. Conclusion：The sensitivity and specificity of FQ-RT-PCR in detecting MDR1 mRNA in bone marrowy cells of children with ALL patients are high. MDR1 mRNA is expressed in children with and without leukemia. MDR1 mRNA is highly expressed in the CR ALL patients with high MRD, recurrence and short duration（within 3 years）. Monitoring MRD and the MDR1 mRNA level might be helpful for individual treatment.

Synchronous Detection of DNA/RNA of Four Shrimp Viruses by Real-time Fluorescence Quantitative RT-PCR

[ Objective] This study aimed to establish a simultaneous detection method of shrimp viruses by real-time fluorescence quantitative RT-PCR, to improve the efficiency of inspection and quarantine. [ Method] A novel real-time fluorescence quantitative RT-PCR assay was established and optimized for simultaneously detecting DNA/RNA of four shrimp viruses （WSSV, IHHNV, TSV and YHV ）. [ Result] The optimized real-time fluorescence quantitative RT-PCR system gener- ated typical amplification curves with high amplification efficiencies （E = 1.06, 1.07, 0.92 and 0.92, respectively）, good hnear relationship （ r = 1 ）, uniform repeatability （ standard deviation = 0.05 - 0.46 ; variation coefficient = 0.26% - 1.62% ） and high sensitivity, exhibiting no significant differences compared with re- al-time fluorescence quantitative PCR （average error of Ct value = 0.04 -0.40; T = 0.53 -2.50; P 〉 0.05 ）. The total detection time was about 1 h. [ Conclusion] The optimized real-time fluorescence quantitative RT-PCR system can be used for rapid detection of WSSV, IHHNV, TSV and YHV.

Identification and Quantitation of Cashmere (Pashmina) Fiber and Wool Using Novel Microchip Based Real-Time PCR Technology

The textile industrial chain all over the world is facing a challenge of differentiating cashmere fiber from mixture of wool and other fibers in case cashmere stocks are adulterated with wool or other fibers. For identification of cashmere in such mixtures, the development of microchip based real-time PCR technology offers a very sensitive, specific, and accurate solution. The technology has been validated with cashmere and wool samples procured from distant farms, and from cashmere goats and sheep of different age and sex. Model samples with incremental raw cashmere or wool content were tested. The experimentally determined content was found to be comparable to the weighed content of the respective fibers in the samples. This technology may prove a cost cutter since it needs only 1.2 μl of the PCR reagent mix. It is substantially faster than traditional real-time PCR systems for being carried as miniature reaction volume in metal microchip. These features allow faster thermal equilibrium and thermal uniformity over the entire array of microreactors. For routine tests or in commercial set up, the microchips are available as ready-to-run with lyophilized reagents in its microreactors to which only 1 μl of the 10-fold diluted isolated DNA sample is added. The lyophilized microchips offer user-friendly handling in testing laboratories and help minimize human error.

Validation of Two Real-Time PCR Approaches for the Relative Quantitation of Pork and Horse DNA in Food Samples

Two real-time PCR methods for the relative quantitation of DNA from meat species in food samples are described: these methods are applicable for horse in processed beef meat products, and pork in raw/processed beef meat products. Test samples were prepared using raw meat admixtures or processed horse/pork in beef food products made to an industry-standard recipe. The methods were subjected to single laboratory method validation, evaluating the performance characteristics of specificity, PCR efficiency and r-squared (r2), Limit of Detection (LOD), Limit of Quantitation (LOQ), and precision and trueness. A limited UK-based inter-laboratory trial of the two methods was completed involving four participating laboratories. Full statistical analysis of the data qualified the applicability of the methods for accurate and sensitive trace-level analysis. The methods were deemed fit for purpose for reproducibly distinguishing between adventitious contamination at 0.1% (w/w), the level for further enforcement action at 1% (w/w), and a level representative of deliberate economically motivated adulteration (10% (w/w)). The data provided evidence that the precision of the two methods was applicable for qualitative and quantitative detection at topically important levels of adulteration. This work has added significant value to the current state of the art in quantitative determination of topical meat species adulteration, allowing analysts to distinguish between adventitious contamination and deliberate adulteration. The resulting methods described in this paper can easily be deployed and used by analytical laboratories for controls and due-diligence testing based on standard laboratory equipment.

The study on quantitative expression of CD44v6mRNA by real-time RT-PCR with the micro-metastases of gastric cancer

Objective： To study the expression of CD44 correlation and the ability of metastasis of tumor cells in gastric carcinoma, and to find the correlation of the quantitative of CD44V6mRNA and the histology expression of CD44v6 in tumors with the clinic-pathologic features, and to make the quantitative expression of CD44v6mRNA. Methods： Twenty patients with gastric carcinoma, 4 patients with gastritis, and 10 apparently healthy controls were recruited. Blood samples were obtained before surgery. 10 days after surgery, the blood samples were obtained again. Serum CIM4v6mRNA in all cases was measured by real-time quantitative PCR. Results： Serum CIM4V6mRNA was detectable in 20 of 20（ 100% ） gastric carcinoma cases, The expression level ranged from 4.9×10^2 copies/μg RNA to 3.2 ×10^5 copies/μg RNA, the average levels of peripheral blood was 3.9 ×10^4 copies/μg RNA, The expression level of pedpheral blood of gastric cancer after curative operation ranged from 5.5×10^0 copies/μg RNA to 7.6 ×10^3 copies/μg RNA. After curative operation the expression level was decreased markedly. Conclusion： Serurn CIM4v6mRNA is expressed in the peripheral blood of gastric carcinoma patients. The expression level of CD44V6mRNA is obviously decreased after curative operation. An elevated level of CD44v6mRNA may serve as an indicator of lymph node metastasis （especially early metastasis） and bad prognosis in patients with gastric carcinoma.

TaqMan real-time fluorescent quantitative RT-PCR in detection of macrophage inflammatory protein-2γ mRNA in myocarditis murine

Objective: To study the role of macrophage inflammatory protein (MIP)-2γ in myocarditis pathogenesis in BALB/c mice. Methods: The relationship between the progression of Coxsarckie virus B3(CVB3) viral myocarditis and experimental autoimmune myocarditis and MIP-2γ mRNA expression in mouse was studied by TaqMan real-time fluorescent quantitative RT-PCR. Results: MIP-2γ mRNA expression rose on 3 to 5 d after CVB3 infection, reached peak on 7 d, and returned to normal level until 14 d, which corresponded well with the disease course. The MIP-2γ mRNA expression level rose significantly on the day 18 d after immunization with porcine cardiac myosin, which was consistent with pathological examination. Conclusion: MIP-2γ may be involved in the pathogenesis of myocarditis.

Real-time Quantitative RT-PCR for CT9 Level in Human Cancer

CT9 is a recently cloned cancer-testis antigen, which is a member of the bromodomain and extraterminal family. Each member of this protein family contains two N-terminal bromodomain motifs. We investigated the distribution of CT9 in different tissues and the possibility for it to be used as a potential therapeutic target in cancer treament. By using the real-time RT-PCR method and 18SrRNA as an internal standard, we analyzed the CT9 expression in several normal human tissues and in the tissues of patients suffering from cancer. The result of this study shows that the highest level of mRNA is only present in testis tissue because the CT9 expression has not been detected in other normal tissues. In 6 of 10 cases of gastric adenocarcinoma, in 3 of 10 cases of esophageal squamous cell carcinoma, in 2 of 9 cases of endometrial carcinoma and only in 1 of 12 cases of brain cancer, the low level expression of CT9 was detected. In none of the 12 cases of cervical squamous cell carcinoma, the expression of CT9 was detected. Since the high level expression of CT9 is only found in the normal testis tissue, but the low expression in cancer tissues, for example tissues of cervical squamous cell carcinoma, brain cancer, endometfial adenocarcinoma, esophageal squamous cell carcinoma, we conclude that CT9 cannot be used as a cancer therapeutic target molecule for cervical squamous cell carcinoma, brain cancer, endometrial adenocarcinoma, esophageal squamous cell carcinoma.

检测粪便中GGⅡ型诺如病毒Real-time RT-PCR方法的建立

诺如病毒是世界急性胃肠炎的重要病原之一。为加强其控制，通过序列比对设计出针对GGⅡ型诺如病毒保守序列的特异性引物与探针，建立了TaqMan Real-time RT-PCR方法。结果显示，此方法对诺如病毒核酸检测高度特异，与轮状病毒、腺病毒、甲型肝炎病毒等无交叉反应，最低检出限可达10^2拷贝，线性范围为100～100拷贝，标准曲线的相关系数为-1．00。针对标准品质粒检测的批内实验变异系数为0．28％～1．63％（n=6）、批间实验为0．28％～1．05％（n=3），对同一样品分6次进行RNA提取和逆转录，其变异系数为3增8％。对212份临床腹泻标本分别用常规RT-PCR和本文建立的TaqMan Real-time PCR进行检测，发现后者检出率略高于前者。这些结果提示此研究建立的诺如病毒的TaqMan Real-time RT-PCR检测方法可用于临床腹泻粪便标本的检测。

利用real-time RT-PCR研究大型蚤对铜绿微囊藻毒素合成基因转录水平影响

近年来关于浮游动物与微囊藻相互作用的研究逐渐被关注,其中有的研究认为浮游动物能够诱导产毒细胞毒素含量的变化.微囊藻毒素是由微囊藻毒素合成基因编码翻译的,目前关于浮游动物对微囊藻毒素合成基因相对表达的影响并无报道,本文首次通过实时定量逆转录PCR方法研究铜绿微囊藻PCC7806产毒相关基因mcyB和mcyD在大型蚤胁迫下相对表达变化.结果显示微囊藻mcyB、mcyD基因相对表达均有上调,表明铜绿微囊藻PCC7806通过上调产毒基因的转录水平达到对大型蚤的诱导防御,从而为浮游动物与微囊藻相互作用研究提供新的依据.

TaqMan MGB Real-time RT-PCR检测西尼罗病毒方法的建立

目的建立一种灵敏、特异、高效的西尼罗病毒(West Nile virus,WNV)感染诊断和流行病学调查的实验室检测方法。方法用C6/36细胞培养WNV并用Vero细胞蚀斑法滴定病毒浓度;根据WNVC蛋白基因组保守序列,设计一套特异性引物和TaqMan MGB探针,用细胞培养病毒液进行方法的条件优化;用不同病毒评价方法的特异性,用系列浓度病毒稀释液和染毒蚊子评价方法的灵敏性。结果建立的TaqMan MGB Real-time RT-PCR方法可检出低于0.01PFU的WNVRNA,并可检出只含3只染毒蚊的标本;用该方法检测4种血清型登革病毒标准毒株、日本脑炎病毒、麻疹病毒、基孔肯亚病毒均为阴性。结论新建TaqMan MGB Real-time RT-PCR方法具有极高的特异性和灵敏性,是实验室早期诊断WNV感染和调查媒介携带WNV情况的理想方法。

猴逆转录病毒RT-PCR和Real-time RT-PCR检测方法的建立

猴逆转录病毒(Simian type D retrovirus,SRV)是引起猴获得性免疫缺陷综合征(Simian acquired immunodeficiency syndrome,SAIDS)的病原之一,其严重危害猴的健康,并威胁与猴接触人员的健康,是无特定病原体(SPF)猴必须排除的病毒之一。为了应对口岸对进出境野生及实验用灵长类动物SRV感染情况的监测和流行病学调查的需要,建立了RT-PCR和real-time RT-PCR检测SRV的方法,并对方法的特异性、敏感性和稳定性进行了验证。

鸡传染性支气管炎病毒Real-time RT-PCR检测方法的建立及应用

为定量检测鸡传染性支气管炎病毒(IBV)载量,建立IBV的Real-timeRT-PCR方法。用RT-PCR方法扩增出IBV的N基因片段,并克隆到pEASY-T3载体中,构建成含有N基因片段的重组质粒。应用该重组质粒进行SYBRGreenⅠReal-timePCR,建立了定量检测IBV核酸的标准曲线与直线回归方程,该方法显示:特异性强,检测下限至少达到5.58×102拷贝/μL,其重复性试验的变异系数小于3.2%;用建立的方法对实验接种IBVM41株的雏鸡组织中的病毒核酸进行了定量检测,检测结果显示:攻毒后肾脏中IBV含量高于支气管和肺脏,支气管和肺脏中病毒含量在攻毒后第3天高于攻毒后第7、10天,并证实临床表现与病毒载量之间存在密切关系。研究结果表明,建立的Real-timeRT-PCR检测方法特异性强、灵敏度高、可重复性好,可用于IBV的定量检测。

Real-time Quantitative PCR Analysis of Vascular Endothelial Growth Factor Receptor-2(VEGFR-2) Expression at Zebrafish Different Developmental Stages

[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2（VEGFR-2） at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish embryos and larvae.Real-time quantitative RT-PCR was performed to examine the expression of VEGFR-2.The data were analyzed by 2^-△△Ct method.[Result]The expression level of VEGFR-2 gene increased gradually from 12 to 72 hpf,and subsequently decreased at 96 hpf.The expression level was lowest at 12 hpf,highest at 72 hpf,and had significant differences when compared with that of other developmental stages.[Conclusion]The expression level of VEGFR-2 increases gradually before blood vessel maturation and decreases as blood vessels mature.

利用Real-time RT-PCR法检测抗病毒活性物质对CMV复制的影响

为准确检测经活性物质处理后的烟草体内病毒含量,建立了real-time RT-PCR检测体系。从烟田中分离到1株对CMV有拮抗作用的细菌B6,经分离纯化得到拮抗CMV的活性物质B6p,其分子量为40.6 kDa。利用叶圆片法,分析了活性物质处理后寄主体内病毒的复制特点,结果表明寄主体内的病毒复制作用受到了拮抗活性物质的抑制,并且随着拮抗活性物质浓度的增加病毒的复制作用减弱。

应用Real-Time RT-PCR鉴定2个水稻品种(品系)对水稻条纹病毒的抗性差异

应用Real-time RT-PCR检测了水稻条纹病毒(Rice stripe virus,RSV)在2种水稻品种(品系)武育粳3号和KT95-418的悬浮细胞内复制变化和相对含量的差异,结合传统生物学接种试验,确定了这2个品种(品系)对RSV抗性的差异.结果表明,RSV在武育粳3号的悬浮细胞内24 h达到复制高峰,病毒含量为侵染初期的7.46倍.而在KT95-418的悬浮细胞内,RSV达到复制高峰需要36 h,病毒含量为侵染初期的4.51倍.利用病毒生物学接种的方法,武育粳3号发病率达91.7%,而KT95-418仅为36.0%.由此可见,KT95-418较武育粳3号对RSV具有较高的抗病性.因此,Real-time RT-PCR方法与传统生物学接种试验方法相比,具有更高的准确性和灵敏性,可以作为传统品种抗病性鉴定的验证手段.

Real-time RT-PCR检测J亚群禽白血病病毒的研究

为了建立检测J亚群禽白血病病毒(ALV-J)的方法,试验采用Real-time RT-PCR,通过1对ALV-J特异性引物对ALV-J山东分离株(命名为ALV-JSD株)进行特异性、敏感性和重复性试验;然后用ALV-JSD株感染1日龄SPF鸡,感染8周后剖检取各组织,用已建立的方法检测ALV-LSD株的感染情况。结果表明:特异性试验中只有以ALV-JSD株cDNA为模板的反应管中能够检测到扩增曲线;敏感性试验测得反应灵敏度达到3.01×1010 copies/μL,比普通PCR灵敏100多倍;对人工感染鸡的不同组织样品进行3次重复检测,病毒检出率为100%;经重复性和实际临床样本检验证实,该方法稳定、可靠,为ALV-J的早期快速诊断建立了特异、灵敏、廉价的定量检测方法。

Real-time fluorescent quantitative PCR非特异性扩增的研究进展

Real-time fluorescent quantitative PCR(RT-qPCR)因其高效快速、操作简便、高度敏感等优点获得广泛应用,但因其强大的放大功能而容易出现非特异性扩增的问题,尤其是荧光染料法中更加明显。文章对RT-qPCR的原理、非特异性扩增的发生因素、解决方案及该技术的应用前景进行了综述。