Real-time RT-PCR For Detection Research Articles

Development of a molecular-beacon-based multi-allelic real-time RT-PCR assay for the detection of human coronavirus causing severe acute respiratory syndrome (SARS-CoV): a general methodology for detecting rapidly mutating viruses

Emerging infectious diseases have caused a global effort for development of fast and accurate detection techniques. The rapidly mutating nature of viruses presents a major difficulty, highlighting the need for specific detection of genetically diverse strains. One such infectious agent is SARS-associated coronavirus (SARS-CoV), which emerged in 2003. This study aimed to develop a real-time RT-PCR detection assay specific for SARS-CoV, taking into account its intrinsic polymorphic nature due to genetic drift and recombination and the possibility of continuous and multiple introductions of genetically non-identical strains into the human population, by using mismatch-tolerant molecular beacons designed to specifically detect the SARS-CoV S, E, M and N genes. These were applied in simple, reproducible duplex and multiplex real-time PCR assays on 25 post-mortem samples and constructed RNA controls, and they demonstrated high target detection ability and specificity. This assay can readily be adapted for detection of other emerging and rapidly mutating pathogens.

Development of the real-time RT-PCR detection system for determination of pandemic influenza A (H1N1) virus

Development of the real-time RT-PCR detection system for determination of pandemic influenza A (H1N1) virus

Multiplex real-time RT-PCR detection of three viruses associated with the bovine respiratory disease complex

Bovine respiratory disease complex (BRDC) is considered the most significant illness associated with feedlot cattle in North America and possibly worldwide. BRDC is a multi-factorial disease with environmental conditions interacting with multiple viral and bacterial pathogens to produce severe respiratory illness. Bovine herpesvirus 1, bovine viral diarrhoea virus and bovine parainfluenza virus 3 are three of the major viruses associated with BRDC. In this study, a multiplex real-time RT-PCR using Taqman primers and probes was developed to detect simultaneously all three of these important BRDC viruses. The assay was optimised and validated using cell-culture infected material and bovine clinical samples from BRDC cases. The sensitivity of the assay was analysed by comparing the multiplex with the individual singleplex assays, which demonstrated that viral detection was not inhibited, by multiplexing the real-time RT-PCRs. This technique is the first reported real-time PCR method for BPIV-3 and the first multiplex designed to detect three BRDC viral pathogens.

Development of a minor groove binder assay for real-time one-step RT-PCR detection of swine vesicular disease virus

The design and development of a 5′ conjugated minor groove binder (MGB) probe real-time RT-PCR assay are described for rapid, sensitive and specific detection of swine vesicular disease virus (SVDV) RNA. The assay is designed to target the 2C gene of the SVDV genome and is capable of detecting 2 × 10 2 copies of an RNA standard per reaction. It does not detect any of the other RNA viruses that cause vesicular disease in pigs, or the human enterovirus, Coxsackie B5 virus (CVB5) which is closely related antigenically to SVDV. The linear range of this test was from 2 × 10 2 to 2 × 10 8 copies/μl. The assay is rapid and can detect SVDV RNA in just over 3.5 h including the time required for nucleic acid extraction. The development of this assay provides a useful tool for the differential diagnosis of SVD or for the detection of SVDV in research applications. This study demonstrates the suitability of MGB probes as a real-time PCR chemistry for the diagnosis of swine vesicular disease.

Analytical performance of norovirus real-time RT-PCR detection protocols in Canadian laboratories

Background Noroviruses (NoVs) are the leading cause of infectious gastroenteritis worldwide. Real-time reverse transcription PCR (real-time RT-PCR) is the preferred method of NoV detection for the majority of testing laboratories. Although the accepted target region for molecular detection assays is the conserved ORF1/ORF2 junction, multiple variations have been published with differences in primers, probes, reagents, multiplexing, etc. Objectives We assessed the detection limit for GII.4 NoV real-time RT-PCR assays as well as the ability to detect the non-GII.4 NoV genotypes in each participating laboratory. Study design A panel of 25 RNA samples was circulated to 18 testing laboratories for comparison of their real-time RT-PCR procedures for NoV detection. Results Multiple protocols with slight differences in reagents or conditions successfully detected 10 genome equivalents or fewer of NoV per reaction. Multiplex procedures were significantly associated ( p = 0.04) with false negative results, particularly for a GI.2 strain. Sensitive detection was associated with false positive results ( p = 0.03). Conclusions Overall, the data indicate that comparable results are produced under slightly different assay conditions.

Detection of carcinoembryonic antigen messenger RNA in blood using quantitative real-time reverse transcriptase-polymerase chain reaction to predict recurrence of gastric adenocarcinoma

BackgroundThe existence of circulating tumor cells (CTCs) in peripheral blood as an indicator of tumor recurrence has not been clearly established, particularly for gastric cancer patients. We conducted a retrospective analysis of the relationship between CTCs in peripheral blood at initial diagnosis and clinicopathologic findings in patients with gastric carcinoma.MethodsBlood samples were obtained from 123 gastric carcinoma patients at initial diagnosis. mRNA was extracted and amplified for carcinoembryonic antigen (CEA) mRNA detection using real-time RT-PCR. Periodic 3-month follow-up examinations included serum CEA measurements and imaging.ResultsThe minimum threshold for corrected CEA mRNA score [(CEA mRNA/GAPDH mRNA) × 106] was set at 100. Forty-five of 123 patients (36.6%) were positive for CEA mRNA expression. CEA mRNA expression significantly correlated with T stage and postoperative recurrence status (P = 0.001). Recurrent disease was found in 44 of 123 cases (35.8%), and 25 of these (56.8%) were positive for CEA mRNA. Of these patients, CEA mRNA was more sensitive than serum CEA in indicating recurrence. Three-year disease-free survival of patients positive for CEA mRNA was significantly poorer than of patients negative for CEA mRNA (P < 0.001). Only histological grade and CEA mRNA positivity were independent factors for disease-free survival using multivariate analysis.ConclusionsCEA mRNA copy number in peripheral blood at initial diagnosis was significantly associated with disease recurrence in gastric adenocarcinoma patients. Real-time RT-PCR detection of CEA mRNA levels at initial diagnosis appears to be a promising predictor for disease recurrence in gastric adenocarcinoma patients.

Influenza A Subtyping: Seasonal H1N1, H3N2, and the Appearance of Novel H1N1

Influenza virus subtyping has emerged as a critical tool in the diagnosis of influenza. Antiviral resistance is present in the majority of seasonal H1N1 influenza A infections, with association of viral strain type and antiviral resistance. Influenza A virus subtypes can be reliably distinguished by examining conserved sequences in the matrix protein gene. We describe our experience with an assay for influenza A subtyping based on matrix gene sequences. Viral RNA was prepared from nasopharyngeal swab samples, and real-time RT-PCR detection of influenza A and B was performed using a laboratory developed analyte-specific reagent-based assay that targets a conserved region of the influenza A matrix protein gene. FluA-positive samples were analyzed using a second RT-PCR assay targeting the matrix protein gene to distinguish seasonal influenza subtypes based on differential melting of fluorescence resonance energy transfer probes. The novel H1N1 influenza strain responsible for the 2009 pandemic showed a melting profile distinct from that of seasonal H1N1 or H3N2 and compatible with the predicted melting temperature based on the published novel H1N1 matrix gene sequence. Validation by comparison with the Centers for Disease Control and Prevention real-time RT-PCR for swine influenza A (novel H1N1) test showed this assay to be both rapid and reliable (>99% sensitive and specific) in the identification of the novel H1N1 influenza A virus strain.

Validation of the Cepheid Xpert Flu A Real Time RT-PCR detection panel for Emergency Use Authorization

Background In April 2009, the United States Secretary of the Department of Health and Human Services declared a public health emergency concerning the 2009 influenza H1N1 outbreak. This declaration allowed the FDA to issue Emergency Use Authorization (EUA) of approved in vitro diagnostics to detect the 2009 influenza H1N1 in clinical specimens. Objectives This report outlines the validation testing of the Cepheid Xpert Flu A Panel for the qualitative detection of 2009 H1N1 viral RNA. Study design This study was a multi-site, dual-method clinical evaluation comparing the results of testing between the Xpert Panel assay to the FDA-cleared Luminex Molecular Diagnostics xTAG™ Respiratory Viral Panel (Luminex RVP) assay and the EUA-granted Focus Diagnostics Influenza A/H1N1 (2009) Real Time RT-PCR (Focus H1N1) assay. Results When compared to Luminex RVP ( n = 300) for influenza A detection, the Xpert Panel had a sensitivity of 91.2% (95% CI: 85.1–95.4), specificity of 99.4% (95% CI: 96.7–100), positive predictive value (PPV) of 99.2% (95% CI: 95.6–100), and a negative predictive value (NPV) of 93.1% (95% CI: 88.3–96.4). When compared to the Focus H1N1 ( n = 258) for detection of H1N1, the Xpert Panel had a sensitivity of 92.1% (95% CI: 82.4–97.4), specificity of 100% (95% CI: 98.5–100), PPV of 100% (95% CI: 95.0–100), and a NPV of 97.5% (95% CI: 94.3–99.2). Conclusions The results show the Cepheid Xpert Flu A Panel to be comparable to both the Luminex RVP and the Focus H1N1 assays. The Cepheid Xpert Panel was granted an EUA on 24 Dec 2009.

A real-time RT-PCR for detection of clade 1 and 2 H5N1 Influenza A virus using Locked Nucleic Acid (LNA) TaqMan probes

BackgroundThe emergence and co-circulation of two different clades (clade 1 and 2) of H5N1 influenza viruses in Vietnam necessitates the availability of a diagnostic assay that can detect both variants.ResultsWe developed a single real-time RT-PCR assay for detection of both clades of H5N1 viruses, directly from clinical specimens, using locked nucleic acid TaqMan probes. Primers and probe used in this assay were designed based on a highly conserved region in the HA gene of H5N1 viruses. The analytical sensitivity of the assay was < 0.5 PFU and 10 - 100 ssDNA plasmid copies. A total of 106 clinical samples (58 from patients infected with clade 1, 2.1 or 2.3 H5N1 viruses and 48 from uninfected or seasonal influenza A virus-infected individuals) were tested by the assay. The assay showed 97% concordance with initial diagnostics for H5 influenza virus infection with a specificity of 100%.ConclusionsThis assay is a useful tool for diagnosis of H5N1 virus infections in regions where different genetic clades are co-circulating.

Implementation of real-time RT-PCR for detection of human metapneumovirus and its comparison with enzyme immunoassay

Human metapneumovirus (hMPV) is responsible for outbreaks of bronchiolitis in winter and early spring in young children. Due to the relatively recent discovery of hMPV, the diagnostic opportunities are limited, while differential diagnosis with respiratory syncytial virus (RSV) remains important. We validated the RT-PCR by comparing various methods of RNA extraction, one-step RT-PCR kits and primer-probe combinations. The optimized RT-PCR was evaluated using 47 nasopharyngeal aspirates (NPAs) collected from children younger than 5 years, with clinically suspected RSV infection. The evaluated RT-PCRs were also compared to a commercially available hMPV enzyme immunoassay (EIA). We found 8.5% hMPV positivity with both RT-PCRs, in agreement with published literature. hMPV EIA showed positive and indeterminate results in 17% and 8.5%, respectively, of the tested NPAs. Positive RT-PCR samples were positive or indeterminate by hMPV EIA. Samples that were positive for RSV and influenza A virus interfered with the hMPV EIA. In conclusion, although RT-PCR is already a valuable tool for diagnosing hMPV infections, further optimization of the RT-PCR method is recommended. The hMPV EIA kit shows poor specificity and therefore needs further improvement.

Prognostic Significance of Real-Time RT-PCR Detection of Disseminated Tumour Cells in Bone Marrow and Circulating Tumour Cells in Patients with Breast Cancer.

Abstract Background: Despite optimal staging and treatment, up to 40% of stage I and II breast cancer (BC) patients will develop recurrent disease over time. These patients are believed to have disseminated tumour cells (DTC) at the time of diagnosis. Detection of minimal disease in bone marrow (BM) has been suggested to be a more direct approach to select metastasis-prone patients among this 'good prognosis group'. Peripheral blood (PB) sampling however is more convenient. The aim of this study was to evaluate whether the detection of DTC in either PB or BM predicts overall survival (OS). The initial analysis was published with a mean follow up time of 786 days (Benoy et al., BrJC 2006). We now report on these data after a mean follow up of nearly 5 years.Material and methods: PB and BM samples were collected from 148 patients with primary (M0, n=116) and metastatic (M+, n=32) BC before the initiation of any local or systemic treatment. PB of healthy volunteers and BM of patients with a nonmalignant breast lesion or a haematological malignancy served as control group. DTC were detected by measuring relative gene expression (RGE) for cytokeratin 19 (CK19) and mammaglobin (MAM), using a quantitative RT-PCR detection method. The 95 percentile of the RGE for CK19 and MAM of the control group was used as cutoff to determine elevation in BC patients. Kaplan-Meier analysis was used to predict OS.Results: Mean follow up time was 1518 days (+/- 719). Elevated CK19 expression was detected in 42 (28%) BM samples and in 22 (15%) PB samples. MAM expression was elevated in 20% (both PB and BM) of the patients with BC. There was a 68% (CK19) and 75% (MAM) concordance between PB and BM samples when classifying the results as either positive or negative. Patients with an elevated CK19 or MAM expression in BM had a worse OS than patients without elevated expression levels (p=0.002 (CK19) and p=0.001 (MAM)). For PB, no statistical significant difference in OS was observed between patients with or without elevated CK19 (p=0.227), but a strong trend for predicting OS was observed according to MAM status (p=0.054). Separate analyses of M0 and M+ patients revealed only a marked difference in OS according to BM CK19 in the M+ patient group. In M0 patients disease free survival (DFS) was not significantly predicted by CK19 and/or MAM status in BM alone (p=0.173 (CK19), p=0.219 (MAM)), but the presence of the double positive phenotype showed a trend for predicting DFS (p=0.059).Conclusions: DTC measured as elevated CK19 or MAM mRNA expression, could be detected in both PB and BM of patients with BC. Only the presence of DTC in BM was highly predictive for OS. However, with longer follow up differences in OS between patients with or without elevated CK19 and particularly MAM expression in PB also tend to become more significant. Furthermore, on the long term, double positivity for CK19/MAM in BM seems to predict DFS in patients with localized BC. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3019.

Laboratory efforts to eliminate contamination problems in the real-time RT-PCR detection of noroviruses

In the current study, laboratory efforts to prevent the presence of positive NTCs (no template controls) during the optimization of a quantitative real-time reverse transcriptase PCR assay for detection of Noroviruses (NoVs) are described. Two DNA types (single-stranded (ss)DNA fragments and plasmid DNA) were used to generate a real-time PCR standard and a high frequency of positive NTCs was noticed in the case of ssDNA fragments. To investigate our suspicion of well-to-well migration of DNA during real-time PCR runs as possible cause of the positive NTCs, an “evaporation-experiment” was set up in which the evaporation of water and the possible co-evaporation of DNA were measured as a function of the DNA type (ssDNA-fragments, plasmid DNA and genomic DNA), the reaction plate seal type (adhesive film or 8-cap strips) and the use of 7 µl of mineral oil as cover layer. Results of this experiment indicated that evaporation of water occurred during real-time PCR runs regardless of the DNA type, the seal type and whether or not 7 µl of mineral oil was used as cover layer. Data from this experiment also suggested co-evaporation of DNA, with an apparent negative correlation between the size of the DNA type and the extent of this co-evaporation. The use of 7 µl of mineral oil as cover layer seemed to prevent to some extent co-evaporation of DNA. The use of plasmids as standard combined with 7 µl of mineral oil as cover layer in the real-time PCR setup resulted in a complete absence of positive NTCs while only minor effects were noticed on the performance of the real-time PCR. In general, our results showed that the high sensitivity of an optimized real-time PCR assay should be considered as – besides a great advantage – a potential risk factor for obtaining false-positive results when using this technique.

Use of a robotic RNA purification protocol based on the NucliSens ® easyMAG™ for real-time RT-PCR detection of hepatitis A virus in bottled water

Hepatitis A virus (HAV) infection is the leading cause of acute viral hepatitis throughout the world. An important part of viral control is rapid detection of HAV in drinking water contaminated with feces. One critical step in HAV detection methods is sample preparation. The objective of this study was to test the efficacy of different approaches to extracting RNA from HAV-inoculated bottled water. The optimal method is based on viral concentration by filtration on membrane filters and elution of adsorbed viruses from filters before RNA extraction and RT-PCR amplification. In this approach, the commercially available NucliSens ® easyMAG™ bio-robot (Biomérieux) performs viral RNA purification with silica magnetic beads, which mediate purification of nucleic acids by binding them and allowing other substances to be washed away. A new rapid simplified NucliSens ® easyMAG™-based approach is described and compared with the classical NucliSens ® easyMAG™ approach and with manual silica-based spin column purifications (Qiagen). The limit of detection (LOD) with the new rapid simplified NucliSens ® easyMAG™ approach was about 1 PFU/1.5 L against about 100 PFU/1.5 L using conventional sample treatments that included a concentration step using ultra-filtration.

Novel H1N1 2009 influenza virus infection in Bangkok, Thailand:effects of school closures

Background: Influenza virus is the major cause of respiratory illness, especially in young and older age groups. Since 1918, many subtypes, defined by hemagglutinine (HA) and neuraminidase (NA), have caused global infections or pandemics. The recently isolated swine origin influenza virus (S-OIV) subtype H1N1 has been defined by the World Health Organization (WHO) as the cause of the present influenza pandemic. Objective: To describe and attempt to predict the epidemiology of the novel H1N1 2009 in Bangkok and to evaluate the effects of school closures during the outbreaks. Materials and methods: The first two human cases infected by this S-OIV subtype H1N1 or H1N1 2009 in Thailand have been reported in May 12, 2009 by the Ministry of Public Health. Between May 12 and July 30, 2009, 1212 nasopharyngeal (NP) swabs from four private hospitals and Chulalongkorn Hospital, Bangkok have been sent to a laboratory for Influenza virus diagnosis. The diagnosis was based on real time RT-PCR for seasonal influenza (H1, H3) and H1N1 2009. Results: One thousand two hundreds and twelve specimens of patients with influenza like illness were tested using real time RT-PCR detection. Between mid June and early July, the number of H1N1 2009 increased rapidly with a high prevalence among the 6- to 20-year olds. School closure policy, long public holiday, and additional preventive measures have led to a rapid reduction in the number of H1N1 2009 positive patients. Conclusion: Preventive measures including school closures are important to slow down the outbreak and thus enable health care centers to cope with the large number of patients. Everyone should play a role in delaying the spread of this pandemic.

Quantification of hepatitis C amino acid substitutions 70 and 91 in the core coding region by real-time amplification refractory mutation system reverse transcription-polymerase chain reaction

Objective. The effects of hepatitis C virus (HCV) sequence variations on the success of antiviral therapy or the development of hepatocellular carcinoma (HCC) are complex for many reasons. Recently, there have been several reports on the effects of genotype 1b HCV core amino acid substitutions 70 and/or 91 on the outcome of antiviral therapies and the clinical course. The purpose of this study was to establish real-time amplification refractory mutation system (ARMS) reverse transcription (RT)-polymerase chain reaction (PCR) assays for easy detection of these HCV mutations. Material and methods. Plasmids p-core-W, including the wild-type HCV core coding region (70R and 91L), and p-core-M, including the mutant-type HCV core (70Q and 91M), were constructed by cloning and PCR-based mutagenesis for control vector of the wild-type core and that of the mutant core, respectively. Using serially diluted forms of these vectors, SyBr Green-based real-time ARMS RT-PCR detection with each of the specific primer pairs was performed. Results. Each primer could clearly distinguish the difference between p-core-W and p-core-M at the same copy numbers. Concerning substitution 70, the ratios 100:1, 10:1, 1:1, 1:10, and 1:100 of p-core-W versus p-core-M could be distinguished, while for substitution 91, the ratios 100:1, 10:1, 1:1, 1:10, 1:100, and 1:1000 could be distinguished, confirming the sensitivity and specificity of the assay. Conclusions. This method could be a useful alternative for the detection of genotype 1b HCV core amino acid substitutions 70 and 91 and be reliably applied for rapid screening.

A SELDI‐TOF MS procedure for the detection, quantitation, and preliminary characterization of low‐molecular‐weight recombinant proteins expressed in transgenic plants

We describe a SELDI-TOF MS procedure for the rapid detection and quantitation of low-molecular-weight recombinant proteins expressed in plants. Transgenic lines of potato (Solanum tuberosum L.) expressing the clinically useful protein bovine aprotinin or the cysteine protease inhibitor corn cystatin II were generated by Agrobacterium tumefaciens-mediated transformation, and then used as test material for the analyses. Real-time RT-PCR amplifications and detection of the recombinant proteins by immunoblotting were first conducted for transformed potato lines accumulating the proteins in different cell compartments. Both proteins were found at varying levels in leaves, depending on their final cellular destination and transgene expression rate. These conclusions drawn from standard immunodetection assays were easily confirmed by SELDI-TOF MS comparative profiling, after immobilizing the leaf proteins of control and transformed lines on protein biochips for weak cationic exchange. This procedure, carried out in less than 2 h, allows for the rapid comparison of recombinant protein levels in transgenic plant lines. The molecular weight of immobilized proteins can also be determined directly from the MS spectra, thus providing a simple way to assess the structural integrity and homogeneity of recombinant proteins in planta, and to identify the most suitable cellular compartments for their heterologous production.

Broadly reactive TaqMan ® assay for real-time RT-PCR detection of rotavirus in clinical and environmental samples

Rotaviruses are enteric pathogens responsible for a significant burden of disease, especially in children, through person-to-person transmission and exposure to contaminated food and water. In the present study, a TaqMan ® probe-based real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay was developed and validated for sensitive and specific detection and quantification of rotavirus for the routine screening of clinical and environmental samples. The assay primers and probes were designed to target the non-structural protein region 3 (NSP3) of rotavirus. The rotavirus real-time RT-PCR assay was found to be specific to rotavirus, but broadly reactive to rotavirus genogroups 1–4, 9, 10 and 12. Specificity testing did not identify any cross-reactivity of the assay with a panel of 36 non-rotavirus enteric virus specimens. The sensitivity of the assay was determined using quantified rotavirus stocks and a plasmid DNA stock. Estimated detection limits in reagent-grade water were five genome equivalent copies (GEC) per reaction and two to four rotavirus particles per reaction. The sensitivity of the assay for detecting rotaviruses in environmental water samples was found to be six virus particles per reaction. The rotavirus real-time RT-PCR assay was effective in detecting rotavirus in all 79 stool specimens obtained from a hospital in India. The results of this study demonstrate that the real-time RT-PCR assay for rotavirus is broadly reactive, specific, and sensitive for detection of rotaviruses in clinical specimens and water samples.

A small subgroup of operable breast cancer patients with poor prognosis identified by quantitative real-time RT-PCR detection of mammaglobin A and trefoil factor 1 mRNA expression in bone marrow

The utility of three different epithelial mRNA markers to detect clinically significant, disseminated tumour cells in bone marrow (BM) was explored. Mammaglobin A (hMAM), trefoil factor 1 (TFF-1) and prostate derived Ets factor (PDEF) mRNA were quantitated by real-time RT-PCR in BM samples from 192 breast cancer patients undergoing surgery (control group: 26 healthy women). During a median follow-up of 72 months, four of the five hMAM BM-positive and three of the seven TFF-1 BM-positive patients experienced a systemic relapse. Kaplan-Meier survival analyses demonstrated significantly shorter recurrence-free-, breast-cancer-specific- and overall survival for both hMAM and TFF-1 BM-positive patients. In contrast, PDEF mRNA quantitation did not reveal any significant differences in the survival analyses. Multivariate Cox regression demonstrated hMAM mRNA BM expression to be an independent predictor of both overall- (hazard ratio = 5.896), breast-cancer-specific- (hazard ratio = 10.208) and systemic-recurrence-free survival (hazard ratio = 14.304). TFF-1 status was related to hMAM status (P < 0.001). Breast cancer patients with pre-operative elevated BM levels of hMAM and/or TFF-1 mRNA seem to constitute a small group of patients with a very poor prognosis.

Mise au point d’une technique de RT-PCR duplex en temps réel pour la détection des virus influenza A et B

Influenza viruses continue to be accountable for winter outbreaks. The potential for developing complications is higher in certain risk groups, such as children, the elderly and individuals with chronic medical conditions. We have presented a duplex real-time RT-PCR for detection of influenza A and B. The sensibility of our method was estimated at 0.01TCID(50)/ml for detection of influenza A and 0.1TCID(50)/ml for influenza B. Any other viruses with respiratory tract tropism were detected with our method. After that, we tested our duplex RT-PCR on 119 samples (nasal aspirates and liquids of broncho-alveolar washes) collected in the CHRU of Lille between November 2005 and January 2006 from patients aged one month to 81 years. Conventional methods such as direct immunofluorescence (IF) assay and/or cell culture were applied on these samples. Four samples were positive for influenza A virus detection and, in particular, one liquid of broncho-alveolar wash for which the direct IF assay was negative. Thus, our method is adapted to diagnosis of flu infections. Nevertheless, our duplex RT-PCR will be tested during a flu outbreak.

Quantitative Real-time RT-PCR Detection for Survivin, CK20 and CEA in Peripheral Blood of Colorectal Cancer Patients

To establish a sensitive method for the early detection of circulating tumor cells (CTCs) in peripheral blood (PB) of colorectal cancer (CRC) patients. PB samples were collected from 156 CRC patients, 40 benign colorectal disease patients, 40 healthy individuals and 45 patients with other solid tumors. The combination of negative and positive immunomagnetic bead method was used to enrich cancer cells. Then, cytokeratin-20 (CK20), survivin and carcinoembryonic antigen (CEA) mRNA were detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). In addition, analyses were carried out for their correlation with patients' clinicopathologic features. The positive rates of survivin, CK20 and CEA mRNA in the PB of CRC patients were 57.7, 47.4 and 39.1%, respectively, and the sensitivity increased from 39.1% of CEA mRNA alone to 60.9% of the combined panel. The expression of the three mRNAs in CRC patients was significantly higher than that in benign control and healthy volunteers, and the expression of survivin and CK20 was not significantly higher than that of patients with other solid tumors. However, the expression of CEA mRNA was significantly higher than that of patients with other solid tumors. The expression of survivin, CK20 and CEA mRNA was significantly correlated with Dukes stages and lymph node metastasis. The combined use of negative and positive immunomagnetic beads followed by amplification of survivin, CK20 and CEA mRNA by means of qRT-PCR is a non-invasive and sensitive assay for the detection of circulating CRC cells. The combined panel improved the sensitivity of detection in CRC patients.