Kinetics of baculovirus replication and release using real-time quantitative polymerase chain reaction

The study of viral-based processes is hampered by (a) their complex, transient nature, (b) the instability of products, and (c) the lack of accurate diagnostic assays. Here, we describe the use of real-time quantitative polymerase chain reaction to characterize baculoviral infection. Baculovirus DNA content doubles every 1.7 h from 6 h post-infection until replication is halted at the onset of budding. No dynamic equilibrium exists between replication and release, and the kinetics are independent of the cell density at the time of infection. No more than 16% of the intracellular virus copies bud from the cell. (C) 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 476-480, 2002; DOI 10.1002/bit.10126.

Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.

Optimisation of a quantitative polymerase chain reaction-based strategy for the detection and quantification of human herpesvirus 6 DNA in patients undergoing allogeneic haematopoietic stem cell transplantation

Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-106 copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.

Quantification of thermotolerant campylobacter in Swiss water treatment plants, by real-time quantitative polymerase chain reaction

A total of 49 wastewater samples from 23 different wastewater treatment plants (WWTPs) were analyzed using real-time quantitative polymerase chain reaction for the presence and quantity of thermotolerant campylobacters. Thermotolerant campylobacters were detected in 87.5% (21/24) and 64% (16/25) of untreated and treated wastewater samples, respectively. Their concentration was sufficiently high to be quantified in 20.4% (10/49) of the samples. In these samples, the concentration ranged from 68 000 to 2292 000 cells/L in untreated wastewater and from 10 800 to 28 000 cells/L in treated water. We conclude that thermotolerant campylobacters present a health hazard for workers at WWTPs in Switzerland. [Authors]

Development and use of a multiplex real-time quantitative polymerase chain reaction assay for detection and differentiation of Porcine circovirus-2 genotypes 2a and 2b in an epidemiological survey.

By the end of 2004, the Canadian swine population had experienced a severe 2 increase in the incidence of Porcine circovirus-associated disease (PCVAD), a problem that was 3 associated with the emergence of a new Porcine circovirus-2 genotype (PCV-2b), previously 4 unrecovered in North America. Thus it became important to develop a diagnostic tool that could 5 differentiate between the old and new circulating genotypes (PCV-2a and -2b, respectively). 6 Consequently, a multiplex real-time quantitative polymerase chain reaction (mrtqPCR) assay that 7 could sensitively and specifically identify and differentiate PCV-2 genotypes was developed. A 8 retrospective epidemiological survey that used the mrtqPCR assay was performed to determine if 9 cofactors could affect the risk of PCVAD. From 121 PCV-2–positive cases gathered for this 10 study, 4.13%, 92.56% and 3.31% were positive for PCV-2a, PCV-2b, and both genotypes, 11 respectively. In a data analysis using univariate logistic regressions, PCVAD compatible 12 (PCVAD/c) score was significantly associated with the presence of Porcine reproductive and 13 respiratory syndrome virus (PRRSV), PRRSV viral load, PCV-2 viral load, and PCV-2 14 immunohistochemistry (IHC) results. Polytomous logistic regression analysis revealed that 15 PCVAD/c score was affected by PCV-2 viral load (P = 0.0161) and IHC (P = 0.0128), but not by 16 the PRRSV variables (P > 0.9); suggesting that mrtqPCR in tissue is a reliable alternative to IHC. 17 Logistic regression analyses revealed that PCV-2 increased the odds ratio of isolating 2 major 18 swine pathogens of the respiratory tract, Actinobacillus pleuropneumoniae and Streptococcus 19 suis serotypes 1/2, 1, 2, 3, 4, and 7, which are serotypes commonly associated with clinical 20 diseases.

Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fluorescent antibody test, quantitative polymerase chain reaction pattern and clinical aspects of rabies virus strains isolated from main reservoirs in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Primer extension based quantitative polymerase chain reaction reveals consistent differences in the methylation status of the MGMT promoter in diffusely infiltrating gliomas (WHO grade II-IV) of adults

Diffusely infiltrating gliomas (WHO grade II-IV) are the most common primary brain tumours in adults. These tumours are not amenable to cure by surgery alone, so suitable biomarkers for adjuvant modalities are required to guide therapeutic decision-making. Epigenetic silencing of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene by promoter methylation has been associated with longer survival of patients with high-grade gliomas who receive alkylating chemotherapy; and molecular testing for the methylation status of the MGMT promoter sequence is regarded as among the most relevant of such markers. We have developed a primer extension-based assay adapted to formalin-fixed paraffin-embedded tissues that enables quantitative assessment of the methylation status of the MGMT promoter. The assay is very sensitive, highly reproducible, and provides valid test results in nearly 100% of cases. Our results indicate that oligodendrogliomas, empirically known to have a relatively favourable prognosis, are also the most homogeneous entities in terms of MGMT promoter methylation. Conversely, astrocytomas, which are more prone to spontaneous progression to higher grade malignancy, are significantly more heterogeneous. In addition, we show that the degree of promoter methylation correlates with the prevalence of loss of heterozygosity on chromosome arm 1p in the oligodendroglioma group, but not the astrocytoma group. Our results may have potentially important implications for clinical molecular diagnosis.

Detecting and resolving position-dependent temperature effects in real-time quantitative polymerase chain reaction

Real-time quantitative polymerase chain reaction (qPCR) depends on precise temperature control of the sample during cycling. In the current study, we investigated how temperature variation in plate-based qPCR instruments influences qPCR results. Temperature variation was measured by amplicon melting analysis as a convenient means to assess well-to-well differences. Multiple technical replicates of several SYBR Green I-based qPCR assays allowed correlation of relative well temperature to quantification cycle. We found that inadequate template denaturation results in an inverse correlation and requires increasing the denaturation temperature, adding a DNA destabilizing agent, or pretreating with a restriction enzyme. In contrast, inadequate primer annealing results in a direct correlation and requires lowering the annealing temperature. Significant correlations were found in 18 of 25 assays. The critical nature of temperature-dependent effects was shown in a blinded study of 29 patients for the diagnosis of Prader-Willy and Angelman syndromes, where eight diagnoses were incorrect unless temperature-dependent effects were controlled. A method to detect temperature-dependent effects by pairwise comparisons of replicates in routine experiments is presented and applied. Systematic temperature errors in qPCR instruments can be recognized and their effects eliminated when high precision is required in quantitative genetic diagnostics and critical complementary DNA analyses.

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

The use of quantitative polymerase chain reaction amplification method to determine sex ratio of human spermatozoa in semen and to determine maternal contamination of amniotic fluid samples

We have modified a technique which uses a single pair of primer sets directed against homologous but distinct genes on the X and Y chromosomes, all of which are coamplified in the same reaction tube with trace amounts of radioactivity. The resulting bands are equal in length, yet distinguishable by restriction enzyme sites generating two independent bands, a 364 bp X-specific band and a 280 bp Y-specific band. A standard curve was generated to show the linear relationship between X/Y ratio average vs. %Y or %X chromosomal content. Of the 51 purified amniocyte DNA samples analyzed, 16 samples showed evidence of high % X contamination while 2 samples demonstrated higher % Y than the expected 50% X and 50% Y chromosomal content. With regards to the 25 processed sperm samples analyzed, X-sperm enrichment was evident when compared to the primary sex ratio whereas Y-sperm was enriched when we compared before and after selection samples.

Minimal residual disease monitoring in multiple myeloma: a comparison between allelic-specific oligonucleotide real-time quantitative polymerase chain reaction and flow cytometry.

BACKGROUND AND OBJECTIVES: Minimal residual disease (MRD) studies are useful in multiple myeloma (MM). However, the definition of the best technique and clinical utility are still unresolved issues. The aim of this study was to analyze and compare the clinical utility of MRD studies in MM with two different techniques: allelic-specific oligonucleotide real-time quantitative PCR (ASO-RQ-PCR), and flow cytometry (FCM). DESIGN AND METHODS: Bone marrow samples from 32 MM patients who had achieved complete response after transplantation were evaluated by ASO-RQ-PCR, using TaqMan technology, and multiparametric FCM. RESULTS: ASO-RQ-PCR was only applicable in 75% of patients for a variety of technical reasons, while FCM was applicable in up to 90%. Therefore, simultaneous PCR/FCM analysis was possible in only 24 patients. The number of residual tumor cells identified by both techniques was very similar (mean=0.29%, range=0.001-1.61%, correlation coefficient=0.861). However, RQ-PCR was able to detect residual myelomatous cells in 17 patients while FCM only did so in 11; thus, 6 cases were FCM negative but PCR positive, all of them displaying a very low number of clonal cells (median=0.014%, range=0.001-0.11). Using an MRD threshold of 0.01% (10(-4)) two risk groups with significantly different progression-free survival could be identified by either PCR (34 vs. 15m, p=0.04) or FCM (27 vs. 10m, p=0.05). INTERPRETATION AND CONCLUSIONS: Although MRD evaluation by ASO-RQ-PCR is slightly more sensitive and specific than FCM, it is applicable in a lower proportion of MM patients and is more time-consuming, while both techniques provide similar prognostic information.

Determination of soybean cultivar resistance to soybean cyst nematode with quantitative polymerase chain reaction

Heterodera glycines, the soybean cyst nematode, is the major pathogen of Glycine max (soybean). Effective management of this pathogen is contingent on the use of resistant cultivars, thus screening for resistant cultivars is essential. The purpose of this research was to develop a method to assess infection of soybean roots by H. glycines with real-time quantitative Polymerase Chain Reaction (qPCR), a prelude to differentiation of resistance levels in soybean cultivars. Two experiments were conducted. In the first one, a consistent inoculation method was developed using to provide active second-stage juveniles (J2). Two-day-old soybean roots were infested with 0 and 1000 J2/mL. Twenty-four hours after infestation, the roots were surface sterilized and DNA was extracted with the DNA FastKit (MP Biomedicals, Santa Ana, CA)). For the qPCR assay, primer pair for single copy gene HgSNO, which codes for a protein involved in the production of vitamin B6, was selected for H. glycines DNA amplification within soybean roots. In the second experiment, compatible Lee 74, incompatible Peking and cultivars with different levels of resistance to H. glycines were inoculated with 0 and 1,000 J2/seedlings. Twenty-four hours post inoculation they were transplanted into pasteurized soil. Subsequently they were harvested at 1, 7, 10, 14 and 21 days post inoculation for DNA extraction. With the qPCR assay, the time needed to differentiate highly resistant cultivars from the rest was reduced. Quantification of H. glycines infection by traditional means (numbers of females produced in 30 days) is a time-consuming practice; the qPCR method can replace the traditional one and improve precision in determining infection levels.

A simplified minimal residual disease polymerase chain reaction method at early treatment points can stratify children with acute lymphoblastic leukemia into good and poor outcome groups

Background Minimal residual disease is an important independent prognostic factor in childhood acute lymphoblastic leukemia. The classical detection methods such as multiparameter flow cytometry and real-time quantitative polymerase chain reaction analysis are expensive, time-consuming and complex, and require considerable technical expertise. Design and Methods We analyzed 229 consecutive children with acute lymphoblastic leukemia treated according to the GBTLI-99 protocol at three different Brazilian centers. Minimal residual disease was analyzed in bone marrow samples at diagnosis and on days 14 and 28 by conventional homo/heteroduplex polymerase chain reaction using a simplified approach with consensus primers for IG and TCR gene rearrangements. Results At least one marker was detected by polymerase chain reaction in 96.4%, of the patients. By combining the minimal residual disease results obtained on days 14 and 28, three different prognostic groups were identified: minimal residual disease negative on days 14 and 28, positive on day 14/negative on day 28, and positive on both. Five-year event-free survival rates were 85%, 75.6%,, and 27.8%, respectively (p<0.0001). The same pattern of stratification held true for the group of intensively treated children. When analyzed in other subgroups of patients such as those at standard and high risk at diagnosis, those with positive B-derived CD10, patients positive for the TEL/AML1 transcript, and patients in morphological remission on a day 28 marrow, the event-free survival rate was found to be significantly lower in patients with positive minimal residual disease on day 28. Multivariate analysis demonstrated that the detection of minimal residual disease on day 28 is the most significant prognostic factor. Conclusions This simplified strategy for detection of minimal residual disease was feasible, reproducible, cheaper and simpler when compared with other methods, and allowed powerful discrimination between children with acute lymphoblastic leukemia with a good and poor outcome.

Real-time Polymerase Chain Reaction Quantification of Porphyromonas gingivalis and Tannerella forsythia in Primary Endodontic Infections

Introduction: Porphyromonas gingivalis and Tannerella forsythia are anaerobic bacteria commonly involved in root canal infections. Although previous investigations have assessed these species by strictly qualitative approaches, accurate determination of their cell levels by a sensitive quantitative technique may contribute with additional information regarding relevance in pain of endodontic origin. Method: The root canal levels of P gingivalis, T forsythia, and total bacteria were investigated by a quantitative polymerase chain reaction (PCR) assay based on unique copy molecular markers. A total of 32 symptomatic (n = 14) and asymptomatic (n = 18) cases of endodontic infections were analyzed. Root canal samples were collected; genomic DNA was extracted and submitted to SYBR Green I real-time PCR targeting the rgpB (P gingivalis), bspA (T forsythia), and rpoB (total bacteria) single copy genes. Results: Overall, R gingivalis, T forsythia, and the coexistence of both species were encountered in 28%, 66%, and 22% of the subjects, respectively. P gingivalis and T forsythia levels ranged from 5.65 x 10(-6) to 1.20 x 10(-2) and from 5.76 x 10(-6) to 1.35 x 10(-1). T forsythia was highly prevalent and numerous in the study groups, whereas P gingivalis was moderately frequent and less abundant, displaying 19-fold lower average levels than the former. Conclusions: The endodontic levels of P gingivalis and T forsythia, individually or in conjunction, did not display significant associations with the manifestation of pain of endodontic origin. (J Endod 2009,35:1518-1524)