One-step Real-time PCR Research Articles

One-step real-time PCR for avian influenza virus RNA detection in hunted wild birds smuggled into Italy: Risk factors and epidemiological implications

One-step real-time PCR for avian influenza virus RNA detection in hunted wild birds smuggled into Italy: Risk factors and epidemiological implications

Abstract 5059: Detection of K-RAS mutation in clinical samples by one-step PNA-mediated real-time PCR clamping techniques

Abstract Mutations in the K-RAS oncogene are frequently found in human cancers. K-RAS mutations may indicate prognosis and drug response, and many new cancer therapies are being targeted to the K-RAS pathway. Many researchers suggest that in colon cancer patients the K-RAS mutation status is strong predictor of resistance to therapy with tyrosine kinase inhibitors such as Erbitux® (cetuximab/ Imclone system Inc) or Vectibix® (panitumumab/ Amgen Inc). Therefore, it is important that detection of K-RAS mutations is fast and simple with high accuracy and high sensitivity for predict drug response and prognosis. To detect a minority of mutant K-RAS among abundant wild-type alleles, we developed a high sensitivity and simple method for detection of K-RAS mutations using PNA-mediated real-time PCR clamping. PNA mediated real-time PCR clamping relies on the following two unique properties of PNA probes. 1) PNA-DNA duplexes generally have greater thermal stability than the corresponding DNA-DNA duplex and 2) PNA oligomers are not recognized by DNA polymerases and consequently can serve as sequence selective clamp during PCR amplification. One-step PNA-mediated real-time PCR clamping method was optimized for detection of K-RAS mutations in exon 12 and 13 with high sensitivity. This method was simple and correct with detection limit approximately 0.1% mutant alleles using 10ng to 50ng of normal DNA as the template. The turnaround time for performing this assay was only 2.5hrs. K-RAS mutations were detected in 24.4% (22/90) of clinical samples, of which 18 had mutations in codon 12 and 4 had these in codon 13. To confirm these results, same clinical samples were analyzed with sequencing assay. The concordant results were obtained from 88 (98%) of the 90 clinical samples by the two assays. The PNA-mediated real-time PCR clamping is a rapid and reliable tool for K-RAS mutation detection and allowed a minority of mutant in clinical field. Keywords: K-RAS, Mutation, PNA, cancer, PCR clamping This work was supported be the Small and Medium Business Administration funded by the Korean Government. (S1060400) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5059.

Decreased mitochondrial DNA content in blood samples of patients with stage I breast cancer

BackgroundAlterations of mitochondrial DNA (mtDNA) have been implicated in carcinogenesis. We developed an accurate multiplex quantitative real-time PCR for synchronized determination of mtDNA and nuclear DNA (nDNA). We sought to investigate whether mtDNA content in the peripheral blood of breast cancer patients is associated with clinical and pathological parameters.MethodsPeripheral blood samples were collected from 60 patients with breast cancer and 51 age-matched healthy individuals as control. DNA was extracted from peripheral blood for the quantification of mtDNA and nDNA, using a one-step multiplex real-time PCR. A FAM labeled MGB probe and primers were used to amplify the mtDNA sequence of the ATP 8 gene, and a VIC labeled MGB probe and primers were employed to amplify the glyceraldehyde-3-phosphate-dehydrogenase gene. mtDNA content was correlated with tumor stage, menstruation status, and age of patients as well as lymph node status and the expression of estrogen receptor (ER), progesterone receptor (PR) and Her-2/neu protein.ResultsThe content of mtDNA in stage I breast cancer patients was significantly lower than in other stages (overall P = 0.023). Reduced mtDNA was found often in post menopausal cancer group (P = 0.024). No difference in mtDNA content, in regards to age (p = 0.564), lymph node involvement (p = 0.673), ER (p = 0.877), PR (p = 0.763), and Her-2/neu expression (p = 0.335), was observed.ConclusionEarly detection of breast cancer has proved difficult and current detection methods are inadequate. In the present study, decreased mtDNA content in the peripheral blood of patients with breast cancer was strongly associated with stage I. The use of mtDNA may have diagnostic value and further studies are required to validate it as a potential biomarker for early detection of breast cancer.

One-step real-time quantitative PCR assays for the detection and field study of Sacbrood honeybee and Acute bee paralysis viruses

Two one-step real-time RT-PCR assays, based on SYBR Green (SG) chemistry, were developed or adapted respectively, for the detection, differentiation, and quantitation of two important honeybee viruses: Sacbrood virus (SBV) and Acute bee paralysis virus (ABPV). Both reactions were optimized to yield the highest sensitivity and specificity. The genome equivalent copies (GEC) detection limit per reaction was 389.3 for the ABPV RT-PCR. The GEC detection limit per reaction was 298.9 for the SBV RT-PCR. Viral detection and identification were confirmed by melting curve analysis and sequencing of the PCR products. Both techniques were used to evaluate Spanish field samples and establish the distribution of these viruses. Acute bee paralysis virus was not detected, and Sacbrood virus was present at low frequencies. The one-step real-time SG RT-PCR methods are fast, accurate, and useful for detecting and quantifying these honeybee viruses, which cause inapparent infections and contribute to the increasing depopulation of honeybee colonies.

Detection of K-ras mutations in azoxymethane-induced aberrant crypt foci in mice using LNA-mediated real-time PCR clamping and mutant-specific probes

Azoxymethane, a rodent colon-specific carcinogen, induce DNA damage, and causes proto-oncogene K-ras point mutations and subsequent tumor formation if DNA damage is not repaired or removed. The present study was designed to detect and characterize K-ras mutations in azoxymethane (AOM)-induced aberrant crypt foci (ACF) in mice, and determine whether dietary supplementation of selenium influences K-ras mutations frequency in ACF using a new PCR technique of locked nucleic acid-mediated real-time PCR clamping combined with mutant-specific probes. K-ras mutations were identified in 33% of AOM-induced ACF. In addition to G to A transition mutation, specific G to T transversion mutation was also identified for the first time in mouse ACF. Furthermore, selenium intake was associated with reduced ACF formation and reduced K-ras mutations rate, respectively, from 112 and 37% in mice fed control diet to 65 and 14% in mice fed selenium-containing diet ( p < 0.05). This is the first report of the use of one-step LNA-mediated real-time PCR clamping to detect K-ras mutations in AOM-induced colon cancer model. It is highly sensitive and can be applied to the detection of early genetic alterations in carcinogen-based animal models.

Novel approach for detecting prohibited species-specific central nervous system tissue contamination in meat by one-step real-time reverse transcriptase PCR.

The dissemination of prohibited species-specific central nervous system (CNS) tissue contamination in meat must be tracked to mitigate human health risk associated with bovine spongiform encephalopathy. The efficiency of compliance monitoring and risk control measures taken by concerned regulatory authorities at meat production facilities to avoid such contamination depends on the ability to detect CNS tissue with a reliable and adequately sensitive quantitative method. A rapid and convenient one-step real-time quantitative reverse transcriptase PCR (qRT-PCR) assay was developed based on the absolute quantification of glial fibrillary acidic protein (GFAP) mRNA as a marker for CNS tissue contamination in meat. The GFAP RNA quantity corresponding to a percentage of CNS tissue in artificially spiked meat was determined using an appropriate in vitro transcribed target GFAP RNA as a calibration standard in the assay. The assay had a linear dynamic range of 10(2) to 10(9) copies of target RNA and was able to detect 0.01% CNS contamination in meat. Further evaluation consisted of an analysis of 272 random meat cuts from carcasses and 109 ground meat samples received from a federally inspected abattoir and two meat processing facilities, respectively, over a 5-month period. The analyzed samples were all negative for CNS tissue contamination at an arbitrarily set lower threshold of 0.025%. Overall, the newly developed one-step qRT-PCR may be useful as an objective quantitative compliance monitoring tool and for setting an acceptable low tolerance threshold for such contamination in meat.

Development of a one-step real-time quantitative PCR assay based on primer-probe energy transfer for the detection of porcine reproductive and respiratory syndrome virus

A one-step real-time RT-PCR method has been developed for the simultaneous detection of both genotypes of porcine reproductive and respiratory syndrome virus (PRRSV). The assay is based on primer-probe energy transfer, and the most important advantage of this is the relative tolerance towards mutations in the target-probe region. The primers and the probe were designed using an alignment of 235 Type 1 (including all subtypes) and Type 2 PRRSV strains. According to the alignment, multiple degenerations were included in the forward and reverse primers to enable the detection of all PRRSV strains deposited in the GenBank. Specificity was tested using 37 different PRRSV strains and eight other swine pathogen viruses. The detection limit was approximately 10 copies of RNA prepared from the Lelystad virus, a European Subtype 3 virus (Belarus strain Soz-8), and an American vaccine virus (Ingelvac MLV ®). One TCID 50 was the detection limit in the case of the cell cultured Lelystad virus and an American wild type isolate, respectively. The melting point analysis revealed melting point decrease, but no significant sensitivity and signal loss in the presence of numerous (up to five) target-probe mismatches, indicating the capability of tolerating even more mutations. The method was suitable for the detection and quantitation of phylogenetically divergent strains and can serve as a robust, high throughput tool for molecular diagnosis of the PRRSV.

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.

Kinetics of WHV-HDV replication in acute fatal course of woodchuck hepatitis

The objective of this study was to evaluate, by developing one-step real-time PCR, the outcome of superinfection with hepatitis D virus (HDV) genotype I in woodchucks that were chronic carriers of woodchuck hepatitis virus (WHV) and did not show relevant signs of liver damage. Three woodchucks (Marmota monax) chronically infected with WHV were superinfected with a woodchuck HDV inoculum. The evolution of the WHV and HDV infections was monitored by quantifying HDV-RNA, WHV-DNA, and HDV-WHV antigens and antibodies. WHV and HDV sequencing was also performed and liver markers were evaluated. Liver damage was assessed using the Ishak method. All woodchucks showed a high HDV viral load, antigenemia and short survival after superinfection. Histopathological examination of autoptic liver samples showed massive liver necrosis compatible with an acute fatal course of hepatitis. The WHV sequencing showed that the virus population was not substituted by the WHV inoculum. The HDV sequencing performed during superinfection and at autopsy indicated amino acid changes in immune dominant regions of the HDV antigen. The strong correlation between acute infection with HDV genotype I and rapid and fatal liver failure indicates that HDV can be an important factor in the prognosis of HDV-WHV-superinfected woodchucks.

Quantitative detection of norovirus excretion in pediatric patients with cancer and prolonged gastroenteritis and shedding of norovirus

Although chronic courses of norovirus infection have been described in immunocompromised patients, little is known about noroviral shedding and correlation with clinical symptoms in these patients. In this report, the quantitative courses of norovirus excretion in nine pediatric patients with hematologic and oncologic disorders and prolonged gastroenteritis were investigated. In a retrospective study multiple fecal samples from nine pediatric cancer patients were examined by a one-step real-time PCR. Clinical data of the patients were reviewed and virological data were correlated with clinical symptoms. All nine patients presented with prolonged illness and prolonged noroviral shedding. Vomiting and diarrhea were associated with high norovirus concentrations and norovirus excretion declined slowly in the patients. Retrospectively, initial PCR-testing for norovirus was performed with a median of 7 days after onset of symptoms. This finding hints at the difficulty of obtaining early diagnosis of the infection in these children. The patients were shedding high norovirus concentration over a long period of time. Results of sequential quantitative PCR-testing for norovirus correlated with clinical symptoms. Both clinical symptoms and quantitative PCR-testings help to define the severity of norovirus infection and to estimate the risk for transmission. To prevent the spread of the disease, usage of virocidal disinfectants and isolation procedures should be maintained as long as patients are positive for noroviruses. Since vomiting is frequent in pediatric patients with oncological conditions, a screening program for rapid detection of norovirus infection in this group of patients should be considered.

Molecular beacon-based real-time PCR method for detection of 15 high-risk and 5 low-risk HPV types

Detection of HPV infections requires a robust time-effective single-step method for efficient screening. A molecular beacon-based one-step multiplex real-time PCR system was developed to detect 15 high-risk (HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and 5 low-risk HPV types (HPV types 6, 11, 42, 43, 44). Molecular beacons detecting high-risk types are 5′-FAM-3′-DABCYL-labelled, molecular beacons for low-risk detection are 5′-TET-3′-DABCYL-labelled, while the internal control added before sample DNA extraction is detected by a 5′-FAM-TexasRed-3′-DABCYL wavelength-shifting molecular beacon. Accordingly, fluorescent data for HPV detection are collected at 530 nm for high-risk types, 560 nm in case of low-risk types and the reaction internal control is detected at 610 nm on a Roche LightCycler 2.0 instrument. The sensitivity for detected types varies between 22 and 700 copies/reaction. The clinical performance was tested on 161 clinical sample DNAs. The MB-RT PCR results were compared to the typing results obtained by the L1F/L1R PCR and hybridization-based system described previously, and the concordance rate between the two systems was 89.44%. The favorable characteristics shown by this multiplex single-step real-time HPV detection system make this promising approach worthy for further development and application for clinical screening.

Development of one-step real-time RT-PCR assay for detection and quantitation of peste des petits ruminants virus

In this study, a rapid and specific TaqMan-based, one-step real-time quantitative reverse transcription PCR (qRT-PCR) has been described for the detection of peste des petits ruminants virus (PPRV). Primers and probe were designed based on the nucleocapsid protein gene sequence. The real-time qRT-PCR assay was able to detect PPRV isolates from very distinct geographical areas (Africa, Middle East and Asia). The specificity of the assay was assessed by including rinderpest virus and other morbillivirus RNAs but none of these tested positive in the assay. The analytical sensitivity of the real-time qRT-PCR assay was achieved through the construction of an in-house PPRV cRNA for the generation of a standard curve. The detection limit of the assay was found to be 8.1 RNA copies per reaction mixture. The assay had excellent intra- and inter-assay reproducibility. In total 30 field samples were screened for the presence of PPRV by conventional RT-PCR in parallel with qRT-PCR. The detection rate increased from 46.7% to 73.3% by use of the real-time qRT-PCR. The real-time qRT-PCR assay described in this report allows the rapid, specific and sensitive laboratory detection of PPRV in tissue samples from field cases.

Expression of nm23-H1 in uveal melanoma

Uveal melanoma (UM) is the most common malignant intraocular tumor in adults. Despite the high accuracy of clinical diagnosis and advances in local treatment, more than 50% of UM patients develop metastasis within 10 years of initial diagnosis. NM23 is one of the human metastasis suppressor genes. Reduced nm23-H1 expression is correlated with high metastatic potential in many different cancers. The purpose of this study is to investigate the expression of nm23-H1 in UM and its potential value as a prognostic marker. Immunostaining of nm23-H1 was verified in five human UM cell lines with different metastatic potentials. The expression level of nm23-H1 mRNA was evaluated with one-step quantitative real-time PCR. The invasion ability of the cell lines was assessed before and after silencing nm23-H1 with small interference RNA. Thirty-two cases of paraffin-embedded specimens of human UM were immunostained with nm23-H1 monoclonal antibody. The immunostaining was evaluated in a semiquantitative fashion based on extent and intensity. The real-time PCR results of five human UM cell lines showed that expression of nm23-H1 was higher in cell lines with low metastatic potential compared with those with high metastatic potential (P<0.05). The invasive ability of the UM cell lines increased after silencing nm23-H1 expression with small interference RNA (P<0.05). The immunostaining of nm23-H1 was cytoplasmic in all cell lines and UM patients samples. The increased immunostaining intensity of nm23-H1 in patients' samples was associated with better survival rate (Kaplan-Meier test P=0.0097). The expression of nm23-H1 was not correlated with other prognostic factors. It can be concluded that nm23-H1 may be a prognostic marker to predict the survival rate of UM patients and it has the potential to identify high-risk patients.

One-step multiplex real-time PCR assay to analyse the latency patterns of Epstein-Barr virus infection

Epstein-Barr virus (EBV) establishes a latent infection with three types of viral gene expression. These latency types can be distinguished by the expression patterns of EBV nuclear antigen (EBNA)1, EBNA2, latent membrane protein (LMP)1, and LMP2. The EBV lytic cycle is initiated by the transcription of the EBV immediate early BZLF1 gene, which can be used to distinguish between a latent and a lytic infection. In this study, a one-step multiplex real-time PCR assay was developed to quantify the EBNA1, EBNA2, LMP1, LMP2, and BZLF1 expression levels simultaneously by relative quantification. To validate this assay, the quantitation of viral gene transcription was performed in EBV-positive B, T, and natural killer cell lines. Because of its rapidity, sensitivity, and specificity, this new assay can be used for quantitative analyses of the latency patterns of EBV infection and the switch from latency to lytic viral replication.

Development of a real-time PCR assay for the diagnosis of scrub typhus cases in India and evidence of the prevalence of new genotype of O. tsutsugamushi

A qualitative syber green real-time PCR with primers designed for a truncated portion of the 56 kDa major outer membrane antigen gene of Orientia tsutsugamushi was used to diagnose scrub typhus from the blood or serum of suspected patients. Sixty-six blood and/or sera samples from fever cases, either with high index of suspicion for scrub typhus and/or positive by Weil–Felix test (≥1:160), were tested with the PCR. Specificity of the PCR was confirmed by end point melt curve analysis and sequencing of the amplicons. A nested PCR for determination of the serotypes of O. tsutsugamushi was performed on to the samples. In real-time PCR strong positive fluorescence was obtained in 73% of the suspected samples. Serotype-specific PCR amplification of some of the positive samples was indicative of the Kuroki type whereas the rest were non-responsive to this test. Sequence analyses of PCR amplicons indicated the presence of new, previously undescribed type of O. tsutsugamushi in this region. This one-step real-time PCR can be used for the detection and confirmation of scrub typhus, when used independently or in conjunction with, the Weil–Felix test, which is still the only available detection test for scrub typhus in most parts of the developing world. Elaborate studies need to be taken up to further evaluate its suitability as specific molecular tool for the diagnosis of scrub typhus and to delineate the prevalent strain types in these regions for a clear epidemiological understanding of this emerging infectious disease.

Decreased expression of P2X 7 in endometrial epithelial pre-cancerous and cancer cells

Objectives To understand the potential role of P2X 7 as biomarker of endometrial cancer, and the molecular mechanisms by which cancerous epithelial cells maintain low expression of P2X 7. Methods Feasibility clinical experimental study. Normal (28), simple or complex hyperplasia (7), complex hyperplasia with atypia (6) and cancer endometrial discarded tissues (40) were obtained from a total of 81 women, ages 25–75. Endpoint for P2X 7 protein was average pixel signal density of tissue immunoreactivity with anti-P2X 7 antibody. Endpoint for P2X 7 mRNA was one-step quantitative Real-Time PCR. Experiments in-vitro included normal (hEVEC) and cancerous cervical epithelial cells (HeLa) transfected with reporter plasmid containing luciferase-3′ untranslated region (3′UTR)-P2X 7 cDNA, using as endpoint steady-state luciferase mRNA levels. Results Levels of P2X 7 protein and mRNA were significantly lower in vivo, in tissues of complex hyperplasia with atypia or endometrial adenocarcinoma, than in tissues of normal endometrium, simple hyperplasia or complex hyperplasia tissues (sensitivity and specificity of 89–100%, p < 0.0001–0.01). Steady-state levels of luciferase mRNA increased over a 6 h incubation period in hEVEC cells transfected with the 3′UTR-P2X 7-luciferase vector, but decreased in HeLa cells transfected with the reporter plasmid. Conclusions Tissue levels of P2X 7 protein and mRNA can differentiate effectively and accurately between normal and benign hyperplastic endometrial tissues from pre-cancerous and cancer tissues. Cancerous epithelial cells degrade P2X 7 mRNA by activation of instability domains located at the 3′UTR of the P2X 7.

An intron-based real-time PCR method for measuring vasopressin gene transcription

The hypothalamus contains distinct neuronal populations that express distinguishing neuropeptides. The supraoptic nucleus contains magnocellular neurons that predominantly express either vasopressin or oxytocin. Transcriptional activators of vasopressin and other neuropeptides have been the subject of much research. Here we present a method of measuring neuropeptide transcription by tailoring one-step quantitative real-time PCR (qRT-PCR) for the analysis of processed and pre-mRNA (heteronuclear RNA). Using moderate and strong hyperosmotic stimuli to induce transcription, we report an increase in vasopressin transcription (pre-mRNA) of 141% and 406% over control levels in response to a 2% injection of 900 mOsm saline or a 1% body weight i.p. injection of 2 M NaCl, respectively. These results agree with a host of studies employing the more labor-intensive method of in situ hybridization histochemistry by which investigators also measured intron-containing heteronuclear RNAs. Furthermore, these results confirm that qRT-PCR with intron-specific primers can be used to rapidly analyze transcription, and suggest an important further benefit of a real-time PCR analysis, such as the ability of measuring transcription of multiple neuropeptides along with other genes from a single sample.

High Copper Selectively Alters Lipid Metabolism and Cell Cycle Machinery in the Mouse Model of Wilson Disease

Copper is essential for human physiology, but in excess it causes the severe metabolic disorder Wilson disease. Elevated copper is thought to induce pathological changes in tissues by stimulating the production of reactive oxygen species that damage multiple cell targets. To better understand the molecular basis of this disease, we performed genome-wide mRNA profiling as well as protein and metabolite analysis for Atp7b-/- mice, an animal model of Wilson disease. We found that at the presymptomatic stages of the disease, copper-induced changes are inconsistent with widespread radical-mediated damage, which is likely due to the sequestration of cytosolic copper by metallothioneins that are markedly up-regulated in Atp7b-/- livers. Instead, copper selectively up-regulates molecular machinery associated with the cell cycle and chromatin structure and down-regulates lipid metabolism, particularly cholesterol biosynthesis. Specific changes in the transcriptome are accompanied by distinct metabolic changes. Biochemical and mass spectroscopy measurements revealed a 3.6-fold decrease of very low density lipoprotein cholesterol in serum and a 33% decrease of liver cholesterol, indicative of a marked decrease in cholesterol biosynthesis. Consistent with low cholesterol levels, the amount of activated sterol regulatory-binding protein 2 (SREBP-2) is increased in Atp7b-/- nuclei. However, the SREBP-2 target genes are dysregulated suggesting that elevated copper alters SREBP-2 function rather than its processing or re-localization. Thus, in Atp7b-/- mice elevated copper affects specific cellular targets at the transcription and/or translation levels and has distinct effects on liver metabolic function, prior to appearance of histopathological changes. The identification of the network of specific copper-responsive targets facilitates further mechanistic analysis of human disorders of copper misbalance.

A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control

BackgroundAvian influenza viruses (AIVs) are endemic in wild birds and their introduction and conversion to highly pathogenic avian influenza virus in domestic poultry is a cause of serious economic losses as well as a risk for potential transmission to humans. The ability to rapidly recognise AIVs in biological specimens is critical for limiting further spread of the disease in poultry. The advent of molecular methods such as real time polymerase chain reaction has allowed improvement of detection methods currently used in laboratories, although not all of these methods include an Internal Positive Control (IPC) to monitor for false negative results.Therefore we developed a one-step reverse transcription real time PCR (RRT-PCR) with a Minor Groove Binder (MGB) probe for the detection of different subtypes of AIVs. This technique also includes an IPC.MethodsRRT-PCR was developed using an improved TaqMan technology with a MGB probe to detect AI from reference viruses. Primers and probe were designed based on the matrix gene sequences from most animal and human A influenza virus subtypes. The specificity of RRT-PCR was assessed by detecting influenza A virus isolates belonging to subtypes from H1–H13 isolated in avian, human, swine and equine hosts. The analytical sensitivity of the RRT-PCR assay was determined using serial dilutions of in vitro transcribed matrix gene RNA. The use of a rodent RNA as an IPC in order not to reduce the efficiency of the assay was adopted.ResultsThe RRT-PCR assay is capable to detect all tested influenza A viruses. The detection limit of the assay was shown to be between 5 and 50 RNA copies per reaction and the standard curve demonstrated a linear range from 5 to 5 × 108 copies as well as excellent reproducibility. The analytical sensitivity of the assay is 10–100 times higher than conventional RT-PCR.ConclusionThe high sensitivity, rapidity, reproducibility and specificity of the AIV RRT-PCR with the use of IPC to monitor for false negative results can make this method suitable for diagnosis and for the evaluation of viral load in field specimens.

Molecular monitoring of chronic phase chronic myeloid leukemia patients treated with 800 mg imatinib

6554 Background: Imatinib mesylate (STI571; Gleevec) is very effective in the therapy of chronic phase chronic myeloid leukemia (CML). Early data suggests that the 800 mg of imatinib may be more effective than 400 mg. However, a little data is available on the role of molecular monitoring in patients receiving 800 mg of imatinib. Methods: We used quantitative reverse transcriptase-polymerase chain reaction (Q-PCR) for monitoring patients enrolled in a non-randomized open-label study of high dose imatinib mesylate in patients with newly diagnosed chronic phase CML. The ABL gene is used as an internal control and the results are reported as a ratio of fusion BCR-ABL/ABL. Results: Data from 56 patients at baseline, 32 pts. at 3 month, 18 pts. at 6 month, and 3 pts. at 9 months was analyzed. The median ratio of BCR-ABL/ABL in bone marrow (BM) was not significantly different from that in peripheral blood (PB). However, significant reduction in levels of BCR-ABL/ABL ratio was noted at 3 moths of therapy (P5 logs. Using a one-step real-time PCR assay, the current data reveals BCR-ABL-negativity in 34% of patients at 3 month, 56% at 6 month and 67% at 9 month. The levels of BCR-ABL transcript at 3 months correlated with Sokal score (R=0.69, P=0.03). Levels of BCR-ABL at all time points correlated with FISH results (R=0.57, P<0.0001). There was also excellent correlation between results obtained using two different laboratories (Quest Diagnostics and Fred Hutchinson Cancer Research Center) (R=0.64, P<0.0001). As an attempt to measure the disease in the entire body rather than in the limited number of cells in the collected sample, we used Q-PCR to measure BCR-ABL transcript in isolated plasma from these samples. BCR-ABL transcript levels from mRNA isolated from plasma and cells showed excellent correlation (R=0.80, P<0.0001). Plasma levels of BCR-ABL were not significantly different from those in cells at baseline, but were significantly higher than cellular levels at 3 months of therapy. Conclusions: This data suggests that Q-PCR provides a useful means for monitoring CML patients being treated with 800 mg imatinab and we believe that long follow-up will help in developing Q-PCR-based algorithm for therapeutic strategy. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Novartis Novartis