SYBR GreenⅠ荧光RT-PCR检测犬瘟热方法的建立和应用

根据GenBank发表的犬瘟热病毒(CDV)的F基因序列,经过分析在F片段的保守区域内设计引物,建立了SYBR GreenⅠ荧光RT-PCR检测CDV的方法,并通过对厦门市宠物医院收集的临床发病和疑似发病的犬病料(包括眼分泌物、鼻拭子、唾液、血液、尿液等)的检测,结果表明,本研究建立的快速检测CDV的SYBR GreenⅠ荧光RT-PCR方法具有特异性强、灵敏度高、操作简便等优点,值得推广应用.     

SYBR Green I荧光RT-PCR检测犬瘟热方法的建立和应用

根据GenBank发表的犬瘟热病毒（CDV）的F基因序列,经过分析在F片段的保守区域内设计引物,建立了SYBR Green I荧光RT-PCR检测CDV的方法,并通过对厦门市宠物医院收集的临床发病和疑似发病的犬病料（包括眼分泌物、鼻拭子、唾液、血液、尿液等）的检测,结果表明,本研究建立的快速检测CDV的SYBR Green I荧光RT-PCR方法具有特异性强、灵敏度高、操作简便等优点,值得推广应用。     

土壤中棉花黄萎病菌SYBR Green Ⅰ荧光RT-PCR定量检测技术研究

为实现田间土壤棉花黄萎病菌的早期检测,建立了土壤中棉花黄萎病菌的SYBR GreenⅠ荧光定量PCR检测方法.以含342bp PCR扩增产物的阳性质粒为参考,构建了标准曲线,并对该曲线的特异性、敏感性、可重复性进行了评价.结果表明,该方法具有快速、特异性强、敏感度高等特点.检测范围在3.8×103-3.8×108cop...     

A Broadly Reactive One-Step SYBR Green I Real-Time RT-PCR Assay for Rapid Detection of Murine Norovirus

A one-step SYBR Green I real-time RT-PCR assay was developed for the detection and quantification of a broad range of murine noroviruses (MNVs). The primer design was based on the multiple sequence alignments of 101 sequences of the open reading frame (ORF)1−ORF2 junction of MNV. The broad reactivity and quantitative capacity of the assay were validated using 7 MNV plasmids. The assay was completed within 1 h, and the reliable detection limit was 10 copies of MNV plasmid or 0.063 median tissue culture infective doses per milliliter of RAW264 cell culture-propagated viruses. The diagnostic performance of the assay was evaluated using 158 mouse fecal samples, 91 of which were confirmed to be positive. The melting curve analysis demonstrated the diversity of MNV in the samples. This is the first report of a broadly reactive one-step SYBR Green I real-time RT-PCR assay for detecting of MNVs. The rapid and sensitive performance of this assay makes it a powerful tool for diagnostic applications.     

大鼠尿激酶SYBR Green I real time RT-PCR引物的设计

设计用于SYBR Green I法实时定量逆转录多聚酶链反应(QRT-PCR)检测大鼠尿激酶型纤溶酶原激活因子(uPA)mRNA的引物。从基因库获取靶基因及相关序列,充分收集争分析相关生物信息学数据,应用Oligo 6.22设计出一对长度为21bp的引物,其GG含量为52.4%;上下游引物3’最稳定二聚体和及发夹结构的能量分别为-1.5、-0.40 kcal/mol和-3.5、-O.90 kcal/mol,引物间最稳定二聚体为-3.1 kcal/mol。5’端和中间△G值较高,高于3’端△G;引发效率分别455和403。实验证明,该引物能够高效、特异地实现对靶序列的检测,适用于SYBR Green I法实时定量检测(uPA)mRNA。     

A one-step RT-PCR assay to detect and discriminate porcine reproductive and respiratory syndrome viruses in clinical specimens

Outbreaks of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) have led to large economic losses and, subsequently, have drawn great attention to its diagnosis and prevention. To facilitate rapid discrimination of HP-PRRSV from classical PRRSV (C-PRRSV), we developed a one-step RT-PCR assay. Primer specificities were evaluated with RNA extracted from 8 viral strains and our results revealed that the primers had a high specificity for PRRSV. The assay sensitivity was 25 copies/μL for both HP-PRRSV and C-PRRSV. A total of 929 serum samples were identified, of which 20.45% were HP-PRRSV-positive and 1.51% were C-PRRSV-positive, which was completely consistent with that of immunochromatochemistry and sequencing method. The proposed assay can detect the virus 2 days prior the onset of symptoms and it can be performed in 2 h, thereby providing a rapid method to discriminate HP-PRRSV from C-PRRSV for the identification and prevention of PRRSV infections.     

Performance of Simplexa Dengue Molecular Assay Compared to Conventional and SYBR Green RT-PCR for Detection of Dengue Infection in Indonesia

Diagnostic tests based on detection of dengue virus (DENV) genome are available with varying sensitivities and specificities. The Simplexa Dengue assay (Focus Diagnostics) is a newly developed real-time RT-PCR method designed to detect and serotype DENV simultaneously. To assess the performance of the Simplexa Dengue assay, we performed comparison with conventional RT-PCR and SYBR Green real-time RT-PCR on patients sera isolated from eight cities across Indonesia, a dengue endemic country. A total of 184 sera that were confirmed using NS1 and/or IgM and IgG ELISA were examined. Using conventional and SYBR Green real-time RT-PCR, we detected DENV in 53 (28.8%) and 81 (44.0%) out of 184 sera, respectively. When the Simplexa Dengue assay was employed, the detection rate was increased to 76.6% (141 out of 184 samples). When tested in 40 sera that were confirmed by virus isolation as the gold standard, the conventional RT-PCR yielded 95% sensitivity while the sensitivity of SYBR Green real-time RT-PCR and Simplexa Dengue assay reached 97.5% and 100%, respectively. The specificities of all methods were 100% when tested in 43 non-dengue illness and 20 healthy human samples. Altogether, our data showed the higher detection rate of Simplexa Dengue compared to conventional and SYBR Green real-time RT-PCR in field/surveillance setting. In conclusion, Simplexa Dengue offers rapid and accurate detection and typing of dengue infection and is suitable for both routine diagnostic and surveillance.     

Development of a broadly reactive nested reverse transcription-PCR assay to detect murine noroviruses, and investigation of the prevalence of murine noroviruses in laboratory mice in Japan

A broadly reactive nested RT-PCR assay to detect MNV was developed and subsequently used to investigate the prevalence of MNV in laboratory mice in Japan. MNV were detected in 8 (22%) of 37 murine stool specimens by second-round PCR, although no positive band was obtained from any specimen by first-round PCR. Genetic analysis of the second round PCR products showed that MNV sequences detected in this study were closely matched (97.2 ∼ 99.1%) to that of MNV-3 (DQ223042). This is the first report demonstrating the prevalence of MNV in Japan.     

A novel, single nucleotide polymorphism-based assay to detect 22q11 deletions

Velocardiofacial syndrome, DiGeorge syndrome, and conotruncal anomaly face syndrome, now collectively referred to as 22q11deletion syndrome (22q11DS) are caused by microdeletions on chromosome 22q11. The great majority ( approximately 90%) of these deletions are 3 Mb in size. The remaining deleted patients have nested break-points resulting in overlapping regions of hemizygosity. Diagnostic testing for the disorder is traditionally done by fluorescent in situ hybridization (FISH) using probes located in the proximal half of the region common to all deletions. We developed a novel, high-resolution single-nucleotide polymorphism (SNP) genotyping assay to detect 22q11 deletions. We validated this assay using DNA from 110 nondeleted controls and 77 patients with 22q11DS that had previously been tested by FISH. The assay was 100% sensitive (all deletions were correctly identified). Our assay was also able to detect a case of segmental uniparental disomy at 22q11 that was not detected by the FISH assay. We used Bayesian networks to identify a set of 17 SNPs that are sufficient to ascertain unambiguously the deletion status of 22q11DS patients. Our SNP based assay is a highly accurate, sensitive, and specific method for the diagnosis of 22q11 deletion syndrome.     

A real-time PCR assay for the detection and quantitation of Campylobacter jejuni using SYBR Green I and the LightCycler

Campylobacter jejuni is recognized as a leading human food-borne pathogen. Traditional biochemical identification for C. jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable (VNC) state. Nucleic acid-based tests have emerged as a useful alternative to traditional testing. In this article, we present fluorescent quantitative PCR assay for quantitative detection of C. jejuni, the assay was carried out using a LightCycler instrument and product formation was monitored continuously with the fluorescent double-stranded DNA binding dye SYBR Green I. When this assay was applied, the assay positive for all of the isolates of C. jejuni tested (11 isolates, including type strain ATCC33560) and negative for all other Campylobacter spp. (three isolates) and several other bacteria (five species tested). The total assay could be completed in 60 min with a detection limit of approximately 1 CFU, and a correlation coefficient was 1.000. Result indicated that fluorescent quantitative detection methods provided a special, sensitive, rapid, reproducible and accurate method for quantitative detection of C. jejuni.     

Microelectronic-sensing assay to detect presence of Verotoxins in human faecal samples

Aims: To develop a novel Vero cell assay that implements a real‐time cell electronic sensing (RT‐CES) system for the determination of the presence of verotoxin‐producing Escherichia coli (VTEC). The assay overcomes the major drawbacks in conventional Vero cell assay, for example, labour‐intensive and time‐consuming. Methods and Results: Cells were grown onto the surfaces of microelectronic sensors that are integrated into the bottom surfaces of the microtiter plate. Cellular viability was monitored in real‐time and quantified based on changes in the sensor’s electrical impedance. For cell viability measurement, the data generated on the RT‐CES system correlated well with those obtained by the Vero cell assay for Verotoxins. To assess cytotoxicity, test cells growing on microelectronic sensors were treated with either supernatant from pure cultures, or stool samples. The specific neutralizing antibodies of VT1 and VT2 were used to identify specific toxins in the samples. Conclusions: The RT‐CES assay provides a sensitive measurement comparable to conventional crystal violet assay. The assay has been successfully and specifically used to identify VTEC in human faecal samples. Significance and Impact of the Study: The RT‐CES assay significantly shortens the testing time from 48 to 72 h required by the crystal violet assay to only 15 h with automated operation.     

A competitive displacement assay to detect saxitoxin and tetrodotoxin

An assay is described which detects saxitoxin (STX) and tetrodotoxin (TTX) by their competitive displacement of [³H]saxitoxin from its receptor in rat brain membranes. The assay has a sensitivity of 0.15 ng STX/ml and 0.8 ng TTX/ml for buffer samples. The assay was also applied to detection of these toxins in unextracted human plasma and found to have a sensitivity of 0.5 ng STX/ml and 0.6 ng TTX/ml. The competitive displacement assay appears to be the most sensitive procedure yet for detection of STX and TTX.     

A quantitative real-time PCR assay for the detection of tetR of Tn10 in Escherichia coli using SYBR Green and the Opticon

Bacteria of implant infections are extremely resistant to antibiotics. One reason for this antibiotic resistance are transposons; the well-known transposon Tn10, for example, mediates tetracycline resistance to Escherichia coli. Two genes of Tn10, tetA and tetR, are essential for the mechanism of resistance. These genes encode a drug-specific efflux protein and a tetracycline repressor protein, respectively. Tn10 is also widely used in molecular biology. For example, tTA, a recombinant derivate of tetR, has been utilised for a highly efficient gene regulation system in mammalian cells. We have examined E. coli isolates from implant infections for tetracycline resistance and for the presence of tetR. A real-time PCR assay was developed for detection of tetR with SybrGreen using the Opticon PCR machine of MJ Research. This method offers a quick, sensitive, efficient, and reliable approach to the detection and quantification of genes. Clinical isolates of E. coli were examined successfully for tetracycline resistance and for the presence of tetR. The real-time PCR is effective using a variety of templates including isolated E. coli DNA, pure colonies, or liquid culture sources. Using quantified standard DNA, this assay can accurately detect as few as 15 copies. Moreover, this assay has the ability to quantify the number of tetR genes in the presence of contaminating mammalian DNA. In conclusion, the tetR real-time PCR offers new methods for detection and quantification of tetracycline-resistant bacteria and tTA in transfected cell-lines or transgenic animals.     

A sensitive and high-throughput assay to detect low-abundance proteins in serum

The ability to detect antigens immunologically is limited by the affinity of the antibodies and the amount of antigens. We have now succeeded in creating a modular, facile amplification system, termed fluorescent amplification catalyzed by T7 polymerase technique (FACTT). Such a system can detect protein targets specifically at subfemtomolar levels ( approximately 0.08 fM). We describe here the detection of Her2 (also known as Neu) from rodent and human sera. FACTT is adaptable to high-throughput screening and automation and provides a practical method to enhance current ELISAs in medical practice.     

GeXP多重PCR技术同时检测12种常见呼吸道病毒

本研究建立了一种基于GeXP多重基因表达遗传分析系统的多重RT-PCR检测方法,该方法可以同时检测12种呼吸道病毒,包括流感病毒A型和B型、季节性H1N1、副流感病毒1～3型、人鼻病毒、人偏肺病毒、腺病毒、呼吸道合胞病毒A型和B型、人博卡病毒。针对病原体保守区序列设计12种病毒的特异性引物,分别用已验证的阳性标本为模板检验多重体系的特异性。多重检测体系在10~3拷贝/μL水平可同时检测到12种病毒。另检测24份临床标本,以real-time RT-PCR为参考标准,进一步验证检测体系。结果表明,这种基于GeXP系统的新方法灵敏度高、特异性强,可以快速同时检测12种常见呼吸道病毒。     

Quantitative RT-PCR assay for VEGF mRNA in human tumors of the kidney

Angiogenesis is the formation of new capillaries from pre-existing vessels, and recent evidence has demonstrated that tumor growth is controlled mainly by angiogenesis. Vascular endothelial growth factor (VEGF) is an endothelium-specific growth factor which is strongly angiogenic in vitro and in vivo. We have developed a quantitative RT-PCR assay for the measurement of VEGF mRNA expression using a real-time procedure based on the use of fluorogenic probes and the ABI PRISM 7700 Sequence Detector System. The assay performance of this method in terms of practicability and reliability is reported with results that seem promising for its widespread use in the clinical laboratory. The method has been applied to the measurement of mRNA of VEGF in human renal cell carcinomas (RCC). Preliminary results show a significantly higher VEGF mRNA expression (ratio values between 181 and 2222) in tumor specimens compared to non-adjacent, non-tumoral tissue of the same subjects.