Competitive Polymerase Chain Reaction Assay Research Articles

The Medawar Prize Acceptance Speech 2022.

The Medawar Prize Acceptance Speech 2022.

Anaplasma ovis Prevalence Assessment and Cross Validation Using Multiparametric Screening Approach in Sheep from Central Tunisia.

We conducted a 5-month-long screening of Anaplasma spp. and Anaplasma ovis infection in sheep from central Tunisia. During this longitudinal study, we investigated the infection dynamics using both direct and indirect assessments validated with a polymerase chain reaction (PCR) as the gold standard method. The experimental design included 84 male lambs aged from 6 to 8 months, and 32 ewes, both chosen randomly from June to November with a periodicity of 2 weeks approximately between June and September, and 1 month between September and November. A total of 9 field visits were carried out in this period during which animals were clinically examined and biological samples were extracted. Thus, a total of 716 blood smears, 698 sera from the nine sampling dates, as well as 220 blood samples from the first and the ninth sampling dates were collected from apparently healthy lambs and ewes, respectively, and analyzed by competitive enzyme-linked immunosorbent assay (cELISA) and polymerase chain reaction (PCR) assay, for the detection of Anaplasma antibodies and A. ovis DNA, respectively. Sera were analyzed by competitive enzyme-linked immunosorbent assay (cELISA) and PCR, for the detection of Anaplasma antibodies and A. ovis DNA, respectively. The Anaplasma spp. initial seroprevalence rate was 33.3% in lambs and 100% in ewes, and it then flowed in an upward trend to reach a maximum of 52.6% in lambs, whereas in ewes, the Anaplasma spp. seroprevalence rate remained unchanged and equal to 100%. Meanwhile, the A. ovis initial molecular prevalence was 22.6% at the first visit and 26.3% at the last visit in lambs, whereas in ewes, the molecular prevalence rates of A. ovis were higher in both the first and the last visit estimated at 100% and 85.7%, respectively. The Kappa coefficient between cELISA and PCR indicated a moderate level of agreement on the first sampling date (0.67) and a low agreement level on the last (0.43). Furthermore, an exploratory data analysis using a multimodal machine learning approach highlighted the underlying pattern of each analytical technique used in this study. In this prospect, we were able to establish the performance of each technique at detecting Anaplasma spp. in sheep. The combination of these approaches should improve the field assessment while promoting a data-based decision in precision epidemiology. The genetic follow-up test relevant to A. ovis msp4 sequences revealed three different genotypes, two of which were previously described in Italy.

Determination of MYD88L265P mutation fraction in IgM monoclonal gammopathies

AbstractWe describe a novel method for the detection of MYD88L265P mutation using a competitive allele-specific polymerase chain reaction (Cast-PCR) assay. This assay has a sensitivity of 1 × 10−3, is applicable in reactions containing very low amounts of DNA (as low as 20 pg), and allowed the detection of MYD88L265P somatic mutation in both tumor-derived DNA (tDNA) and cell-free DNA (cfDNA). In addition, using the Cast-PCR assay, we were able to determine the mutation allele fraction (MAF) in each tested sample. We then analyzed baseline tDNA and cfDNA samples from 163 patients (53 with immunoglobulin M monoclonal gammopathy of undetermined significance and 110 with Waldenström's macroglobulinemia [WM], of whom 54 were asymptomatic and 56 were symptomatic) and also in sequential samples of 37 patients. MAF in both cfDNA and tDNA was higher among patients with symptomatic compared with asymptomatic WM and in those with asymptomatic WM compared with those with immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance. In addition, the evaluation of sequential samples showed that MAF decreased after treatment, whereas it increased in patients who relapsed or progressed to symptomatic WM. Thus, Cast-PCR is a highly sensitive, cost-effective diagnostic tool for MYD88L265P detection, applicable in both tDNA and cfDNA samples, that also provides a quantitative evaluation of the tumor load in patients with IgM monoclonal gammopathies.

CCL3L3-null status is associated with susceptibility to systemic lupus erythematosus

The correlation between copy number variation (CNV) and the susceptibility to systemic lupus erythematosus (SLE) has been reported for various immunity-related genes. However, the contribution of CNVs to SLE susceptibility awaits more investigation. To evaluate the copy numbers in immunity-related genes such as TNFAIP3, TNIP1, IL12B, TBX21 (T-bet), TLR7, C4A, C4B, CCL3L1, and CCL3L3, the modified real competitive polymerase chain reaction (mrcPCR) assay was employed, and the association between the copy numbers and SLE susceptibility was analyzed in 334 SLE patients and 338 controls. CCL3L3-null status was significantly associated with SLE susceptibility (OR > 18, P < 0.0001), which remained significant by Bonferroni’s correction (corrected P = 0.0007). However, the significant association between C4B low-copy status and SLE susceptibility (OR = 1.6051, P = 0.0331) became non-significant by Bonferroni’s correction (corrected P = 0.3938). Except for these results, no other significant association between SLE susceptibility and copy number status in other genes was observed. The CCL3L3-null status may be a significant factor for SLE susceptibility.

Blood group genotyping of blood donors: validation of a highly accurate routine method.

In the past century, blood group determination using serology has been the standard method. Now, molecular methods are gaining traction, which provide additional and easily accessible information. Here we designed and validated a high-throughput extended genotyping setup. We developed 35 competitive allele-specific polymerase chain reaction assays for genotyping of blood donors. Samples from 1034 Danish blood donors were genotyped, and 45,314 red blood cell antigens and 6148 platelet antigens were predicted. Predicted phenotypes were compared with 16,119 serologic phenotypes. We found 62 discrepancies of which 43 were due to serology. After exclusion of the discrepancies caused by serology, the accuracy of genotyping was 99.9%. Of 17 discrepancies caused by the genotype, three were incorrect antigen-negative predictions and could potentially, as the solitary analysis, have caused an adverse transfusion reaction. We have established a robust and highly accurate blood group genotyping system with a very high capacity for screening blood donors. The system represents a significant improvement over the former serotyping-only procedure. Almost all new technology in medicine incurs increased costs, but the presented efficient genotyping system is a rare example of a significant qualitative and quantitative technologic progress that is also more cost-efficient than previous technologies.

Implementation of next generation sequencing technology for somatic mutation detection in routine laboratory practice

The introduction of next generation sequencing (NGS) in the routine diagnostic setting is still in the development phase and has been limited by its complexity. Targeted NGS offers an attractive alternative to performing multiple single target assays and is very useful in meeting the increasing clinical demand for testing of multiple genetic aberrations in cancer specimens. To this end, we carried out a blinded validation study on 113 tumours in a diagnostic laboratory and compared mutation results from targeted NGS with those from Sanger sequencing, pyrosequencing, competitive allele specific TaqMan polymerase chain reaction (CAST PCR) and Cobas assays. DNA was extracted from formalin fixed, paraffin embedded (FFPE) tissue samples that included core biopsies, resections and cytology samples from three common and one rare cancer types [non-small cell lung cancer (NSCLC), colorectal cancer (CRC), malignant melanoma (MM) and gastrointestinal stromal tumour (GIST)]. Libraries were prepared using the TruSight Tumour 26 gene panel and NGS was carried out on the MiSeq instrument. Results from NGS were concordant with the mutational status determined by other platforms in 107 of the 113 cases tested (94.7%). The sequencing quality for NGS failed in four of the six false negative cases, while a further two samples gave false negative results because the c-KIT mutations were located outside the range of the NGS panel. One NSCLC sample contained an EGFR mutation previously detected by the Cobas assay. Reanalysis of the NGS data for this sample using a cut-off allele frequency of 1% revealed the mutation had an allele frequency of 2%, which was below the recommended software-determined threshold of 3%. NGS detected 113 additional mutations that were not previously known from analysis by the conventional methods. Twenty-six of these have known clinical importance, 37 have potential clinical significance, while 50 were novel mutations with unknown clinical significance. NGS detected variants using inputs of 10-20ng of FFPE extracted DNA and from specimens with a tumour cell content less than 50%, for which when possible we recommend microdissection. We conclude that results from targeted NGS are highly concordant with those from other mutation testing platforms. Targeted NGS is suitable for a range of sample types received in the diagnostic pathology laboratory, including those with limited material or with low tumour cell content (TCC). This work has allowed us to determine the quality parameter settings required in order to obtain robust mutation data by NGS.

The association of novel polymorphisms with stress fracture injury in Elite Athletes: Further insights from the SFEA cohort

ObjectivesTo determine, in conjunction with a wider investigation, whether 11 genetic variants in the vicinity of vitamin D, collagen and Wnt signalling pathways were associated with stress fracture injury in the Stress Fracture Elite Athlete (SFEA) cohort. DesignGenotype-phenotype association study. MethodsSelf-reported stress fracture history and demographic data were recorded in 518 elite athletes, 449 male and 69 female (mean age 24.2±5.5 years) from the SFEA cohort. Elite athletes were assigned to two groups based on history of stress fracture injury. Data were analysed for the whole cohort and sub-stratified in to male only and multiple stress fracture cases. Genotype was determined using a proprietary fluorescence-based competitive allele-specific polymerase chain reaction assay. ResultsSOST SNP rs1877632 and VDR SNPs rs10735810 and rs731236 were associated with stress fracture (p<0.05). In the whole cohort, rs1877632 heterozygotes and homozygotes of the rare allele combined made up 59% of stress fracture sufferers in comparison to 46% in the non-stress fracture group (p=0.05). In the multiple stress fracture cohort, homozygotes of the rare allele of rs10735810 and rs731236 showed an association with stress fracture when compared to those homozygotes for the common allele combined with heterozygotes (p=0.03; p=0.01). No significant associations were shown in the other SNPs analysed (p>0.05). ConclusionsThese data suggest an important role for SOST SNP rs1877632 and VDR SNPs rs10735810 and rs731236 in the pathophysiology of stress fracture. This might be due to the role of the SNPs in the regulation of bone remodelling and adaptation to mechanical loading, with potential implications for the prevention and treatment of stress fracture injuries.

Vitamin D status, serum lipid concentrations, and vitamin D receptor (VDR) gene polymorphisms in Familial Mediterranean fever.

Vitamin D (VitD) is critical for the regulation of inflammatory processes, and VitD deficiency has been linked to several chronic inflammatory disorders. We aimed to investigate the concentrations of serum 25(OH)D3, lipid parameters, and three known VDR polymorphisms (BsmI, FokI, and TaqI) in patients with Familial Mediterranean fever (FMF), an autosomal recessive autoinflammatory disease. The study included 123 FMF patients and 105 controls. Seventy patients had no attack (group 1), 30 had 1-2 attacks (group 2), and 23 had 3 or more attacks (group 3) within last three months. Serum 25(OH)D3 concentrations were determined using liquid chromatography-tandem mass spectrometry. BsmI, FokI, and TaqI polymorphisms were analyzed by a competitive allele specific polymerase chain reaction assay (KASPar). Serum lipid parameters were measured with enzymatic colorimetric methods. 25(OH)D3 concentrations were lower in FMF patients compared to controls (p < 0.001). No difference was observed in 25(OH)D3 concentration between groups 1, 2, and 3. The distributions of FokI and TaqI genotypes were not significantly different between FMF patients and controls. There was a significant difference in the distribution of AA BsmI genotype between male FMF patients and male controls. Increased concentrations of triglycerides (p = 0.012) and decreased concentrations of high-density lipoprotein cholesterol [HDL-C] (p = 0.006) were found in FMF patients compared to controls. Although lower 25(OH)D3 concentrations were observed in FMF patients versus controls, no association was determined between FMF attack frequency and 25(OH)D3 concentrations. We showed that the AA genotype of BsmI polymorphism is associated with FMF in males but not in females. The effects of decreased HDL-C and increased triglyceride concentrations on cardiovascular events in FMF patients should be further investigated.

Markers Linked to Wheat Stem Rust Resistance Gene Sr11 Effective to Puccinia graminis f. sp. tritici Race TKTTF.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici, can cause severe yield losses on susceptible wheat varieties and cultivars. Although stem rust can be controlled by the use of genetic resistance, population dynamics of P. graminis f. sp. tritici can frequently lead to defeat of wheat stem rust resistance genes. P. graminis f. sp. tritici race TKTTF caused a severe epidemic in Ethiopia on Ug99-resistant 'Digalu' in 2013 and 2014. The gene Sr11 confers resistance to race TKTTF and is present in 'Gabo 56'. We identified seven single-nucleotide polymorphism (SNP) markers linked to Sr11 from a cross between Gabo 56 and 'Chinese Spring' exploiting a 90K Infinium iSelect Custom beadchip. Five SNP markers were validated on a 'Berkut'/'Scalavatis' population that segregated for Sr11, using KBioscience competitive allele-specific polymerase chain reaction (KASP) assays. Two of the SNP markers, KASP_6BL_IWB10724 and KASP_6BL_IWB72471, were predictive of Sr11 among wheat genetic stocks, cultivars, and breeding lines from North America, Ethiopia, and Pakistan. These markers can be utilized to select for Sr11 in wheat breeding and to detect the presence of Sr11 in uncharacterized germplasm.

Genetic Associations with Changes in Bone Phenotypes in Academy Soccer Players

The high magnitude, frequency and multi-directional nature of the movements that soccer necessitates are likely to be key to the osteogenic effects of participation. Despite soccer having anabolic effects on bone, stress fracture is a common injury in elite players. Genotype may mediate the bone response to exercise and explain some of the variability in the bone adaptations that occur. PURPOSE: To determine whether eight SNPs in the proximity of the RANK/RANKL/OPG and Wnt signalling pathways were associated with changes in bone phenotypes following 12 weeks of increased training volume in academy soccer players. METHOD: Eighty male players (age 16±0 y; height 1.76±0.07 m; body mass 70.3±8.1 kg) gave written informed consent to take part in a study approved by the National Research Ethics Service. Peripheral quantitative computed tomography scans of the tibia were performed before and 12 weeks after an increase in training volume. Scans were obtained at 4%, 14%, 38% and 66% of tibial length. Trabecular density (mg/cm3), cortical density (mg/cm3), cortical area (mm2), cortical thickness (mm) and strength strain index (mm3) were assessed. Soccer players were genotyped using proprietary fluorescence-based competitive allele-specific polymerase chain reaction assays (LGC genomics Herts, UK). RESULTS: Five SNPs were associated with changes to bone phenotypes following increased training volume (P<0.05). SNPs (RANKL rs9594759; rs1021188; rs9594738) in the proximity of the RANK/RANKL/OPG signalling pathway were associated with increased cortical density (18.5mg·cm3, 1.7%), cross-sectional area (16.6mm2, 7.4%) and thickness (6.7mm, 6.7%). SNPs (Wnt16 rs2707466; SOST rs1877632) in the proximity of the Wnt signalling pathway were associated with increased trabecular density (17.4mg·cm3, 6.1%). CONCLUSIONS: The association of SNPs in the proximity of the RANK/RANKL/OPG and Wnt signalling pathways with bone phenotypes suggests that these SNPs may be important in governing skeletal responses to exercise. It also highlights that genetic mediation of bone formation and resorption pathways are likely to influence bone adaptations in response to exercise. Knowledge related to the genetic governance of bone phenotypic adaptions has the potential to influence exercise prescribed in order to improve bone health.

Intra-tumor heterogeneity of BRAF V600E mutation in lung adenocarcinomas.

BRAF mutations exist in numerous types of cancer, including melanomas, colorectal cancers and lung cancers. The V600E-specific inhibitor vemurafenib has marked clinical activity in patients with BRAF V600E-mutated melanoma. However, there are many cases of resistance to vemurafenib. This may be due to the reported intra-tumor heterogeneity of the BRAF V600E mutation in primary melanomas. BRAF mutations are found in 1-5% of non-small cell carcinomas (NSCLCs), almost exclusively in adenocarcinoma. A few cases have been reported in which vemurafenib was effective against BRAF V600E-mutated lung cancers. In a previous study, five lung adenocarcinomas with BRAF V600E mutation were detected by direct sequencing. The present study analyzed these tumors for the percentage of mutation (%mutation) by competitive allele-specific polymerase chain reaction (CAST-PCR) assay. In addition, sections of all components of the adenocarcinomas were obtained by laser microdissection and analyzed. The %mutations of BRAF V600E within the macrodissected tumors (cases 1-5) were: Case 1, 10.0%; case 2, 8.0%; case 3, 8.9%; case 4, 21.5%; and case 5, 14.9%. In four cases (cases 2-5), the %mutations of each adenocarcinoma component were as follows: Case 2, lepidic growth 6.5-24.5%, papillary 1.3-11.2% and acinar 9.8%; case 3, solid 2.5-69.9%, acinar 12.4-27.1% and papillary 3.7-17.4%; case 4, acinar 10.0-45.0% and papillary 44.0%; and case 5, papillary 3.7-93.4%. Sensitive BRAF mutation detection methods were used and evidence for heterogeneity of the BRAF V600E mutation in these lung adenocarcinoma cases was observed. Targeted therapy with a BRAF V600E inhibitor such as vemurafenib may have potential in the treatment of lung cancer with this mutation; however, it is necessary to consider how the treatment effect of and drug resistance to BRAF V600E inhibitors are affected by the presence of heterogeneity in future studies.

Hormone-sensitive lipase – quantitation of enzyme and mRNA in small biopsies of human adipose tissue

Hormone-sensitive lipase (HSL) activity was first identified as an adrenaline-sensitive lipolytic activity in adipose tissue. Hormonal activation of HSL occurs via cAMP-dependent protein kinase, which phosphorylates HSL. HSL is the enzyme responsible for the release of free fatty acids (FFAs) from adipose tissue and, therefore, plays a pivotal role in providing the major source of energy for most tissues. Although its expression is highest in adipose tissue, HSL is also expressed in the adrenal glands, ovaries, testes and, to a lesser extent, in skeletal and cardiac muscle and macrophages. The aim of this article is to describe a modified method of determination of HSL activity and gene expression in 10 mg of frozen human adipose tissue samples obtained by biopsy. The enzymes were extracted from the frozen tissue and activity was tested against a fatty acid-labelled diacylglycerol emulsion. The released fatty acids were extracted using a liquid–liquid partition system. The mean±standard error of the mean (SEM) HSL activity in normal fasting individuals was 2.38±0.75 milliunits (mU) oleate/min/g at 37°C. HSL mRNA levels were assessed in 10 mg of adipose tissue using a competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay and the mean ratio of HSL mRNA molecules to β2-microglobulin-mRNA molecules in normal individuals was 0.29±0.04 (mean±SEM). The methods described here are suitable for use in adipose tissue biopsies obtained from normal individuals and provide a means for the examination of the regulation of this enzyme. The importance of this is becoming increasingly apparent, as the inability to suppress lipolysis is associated with conditions of insulin resistance and an increase in propensity to atherosclerosis. This is associated with inappropriate release of FFAs in the postprandial period, resulting in reduced sensitivity of glucose metabolism and increased postprandial lipaemia.

Large-Scale Development of Cost-Effective Single-Nucleotide Polymorphism Marker Assays for Genetic Mapping in Pigeonpea and Comparative Mapping in Legumes

Single-nucleotide polymorphisms (SNPs, >2000) were discovered by using RNA-seq and allele-specific sequencing approaches in pigeonpea (Cajanus cajan). For making the SNP genotyping cost-effective, successful competitive allele-specific polymerase chain reaction (KASPar) assays were developed for 1616 SNPs and referred to as PKAMs (pigeonpea KASPar assay markers). Screening of PKAMs on 24 genotypes [23 from cultivated species and 1 wild species (Cajanus scarabaeoides)] defined a set of 1154 polymorphic markers (77.4%) with a polymorphism information content (PIC) value from 0.04 to 0.38. One thousand and ninety-four PKAMs showed polymorphisms between parental lines of the reference mapping population (C. cajan ICP 28 × C. scarabaeoides ICPW 94). By using high-quality marker genotyping data on 167 F2 lines from the population, a comprehensive genetic map comprising 875 PKAMs with an average inter-marker distance of 1.11 cM was developed. Previously mapped 35 simple sequence repeat markers were integrated into the PKAM map and an integrated genetic map of 996.21 cM was constructed. Mapped PKAMs showed a higher degree of synteny with the genome of Glycine max followed by Medicago truncatula and Lotus japonicus and least with Vigna unguiculata. These PKAMs will be useful for genetics research and breeding applications in pigeonpea and for utilizing genome information from other legume species.

Identification of translocation products but not K-RAS mutations in memory B cells from patients with multiple myeloma

Several laboratories have shown that cells with a memory B-cell phenotype can have the same clonotype as multiple myeloma tumor cells. The aim of this study was to determine whether some memory B cells have the same genetic alterations as their corresponding multiple myeloma malignant plasma cells. The methodology included sorting multiple myeloma or memory B cells into RNA stabilizing medium for generation of subset-specific polymerase chain reaction complementary DNA libraries from one or 100 cells. Cells with the phenotype of tumor plasma cells (CD38(++)CD19(-)CD45(-/+)CD56(-/+/++)) or memory B cells (CD38(-)/CD19(+)/CD27(+)) were isolated by flow activated cell sorting. In samples from all four patients with multiple myeloma and from two of the three with monoclonal gammopathy of undetermined significance, we identified memory B cells expressing multiple myeloma-specific oncogenes (FGFR3; IGH-MMSET; CCND1 high) dysregulated by an IGH translocation in the respective tumor plasma cells. By contrast, in seven patients with multiple myeloma, each of whom had tumor plasma cells with a K-RAS61 mutation, a total of 32,400 memory B cells were analyzed using a sensitive allele-specific, competitive blocker polymerase chain reaction assay, but no K-RAS mutations were identified. The increased expression of a specific "early" oncogene of multiple myeloma (monoclonal gammopathy of undetermined significance) in some memory B cells suggests that dysregulation of the oncogene occurs in a precursor B-cell that can generate memory B cells and transformed plasma cells. However, if memory B cells lack "late" oncogene (K-RAS) mutations but express the "early" oncogene, they cannot be involved in maintaining the multiple myeloma tumor, but presumably represent a clonotypic remnant that is only partially transformed.

Development and use of quantitative competitive PCR assays for relative quantifying rumen anaerobic fungal populations in both in vitro and in vivo systems

This paper describes the use of a quantitative competitive polymerase chain reaction (QC-PCR) assay; using PCR primers to the rRNA locus of rumen fungi and a standard-control DNA including design and validation. In order to test the efficiency of this method for quantifying anaerobic rumen fungi, it has been attempted to evaluate this method in in vitro conditions by comparing with an assay based on measuring cell wall chitin. The changes in fungal growth have been studied when they are grown in in vitro on either untreated (US) or sodium hydroxide treated wheat straw (TS). Results showed that rumen fungi growth was significantly higher in treated samples compared with untreated during the 12 d incubation ( P < 0.05) and plotting the chitin assay's results against the competitive PCR's showed high positive correlation ( R 2 ≥ 0.87). The low mean values of the coefficients of variance in repeatability in the QC-PCR method against the chitin assay demonstrated more reliability of this new approach. And finally, the efficiency of this method was investigated in in vivo conditions. Samples of rumen fluid were collected from four fistulated Holstein steers which were fed four different diets (basal diet, high starch, high sucrose and starch plus sucrose) in rotation. The results of QC-PCR showed that addition of these non-structural carbohydrates to the basal diets caused a significant decrease in rumen anaerobic fungi biomass. The QC-PCR method appears to be a reliable and can be used for rumen samples.

DIAGNOSING DIFFERENT STAGES OF HEPATITIS B INFECTION USING A COMPETITIVE POLYMERASE CHAIN REACTION ASSAY

Purpose: Different stages of hepatitis B virus (HBV) infection can be defined by serum HBV DNA levels. This study attempts to (1) investigate serum HBV DNA levels in inactive carriers and patients with chronic HBV (CHB) infection and (2) define cut-off value between inactive carriers and HBeAg (precore antigen of HBV) negative CHB patients in Indian population. Methods: One hundred and forty samples encompassing 42 inactive HBsAg carriers and 98 CHB patients (53 HBeAg-positive and 45 HBeAg-negative) were analysed. Serum HBV DNA levels were determined employing an in-house competitive polymerase chain reaction (cPCR) assay. Results: The HBeAg-positive patients were found to have the maximum median HBV DNA load, which was significantly higher than the HBeAg-negative ones (median; 1.25 ×108vs. 2.30 ×105copies/mL; P < 0.05). Interestingly, the latter group has significantly higher HBV DNA levels than the inactive carriers (median; 2.30 ×105vs. 4.28 ×103copies/mL; P < 0.05). The 2.5 ×104copies/ml HBV DNA levels were optimal for discriminating CHB patients (HBeAg-negative) from inactive carriers with 75.6 and 78.6% sensitivity and specificity, respectively. Conclusions: Despite the extensive overlapping of HBV DNA levels in inactive carriers and HBeAg negative CHB patients, 2.5 ×104copies/mL is the most favourable cut-off value to classify these individuals and would be imperative in the better management of this dreadful disease.

Diagnosing different stages of hepatitis B infection using a competitive polymerase chain reaction assay

Different stages of hepatitis B virus (HBV) infection can be defined by serum HBV DNA levels. This study attempts to (1) investigate serum HBV DNA levels in inactive carriers and patients with chronic HBV (CHB) infection and (2) define cut-off value between inactive carriers and HBeAg (precore antigen of HBV) negative CHB patients in Indian population. One hundred and forty samples encompassing 42 inactive HBsAg carriers and 98 CHB patients (53 HBeAg-positive and 45 HBeAg-negative) were analysed. Serum HBV DNA levels were determined employing an in-house competitive polymerase chain reaction (cPCR) assay. The HBeAg-positive patients were found to have the maximum median HBV DNA load, which was significantly higher than the HBeAg-negative ones (median; 1.25 x 10(8) vs. 2.30 x 10(5) copies/mL ; P<0.05). Interestingly, the latter group has significantly higher HBV DNA levels than the inactive carriers (median; 2.30 x 10(5) vs. 4.28 x 10(3) copies/mL; P<0.05). The 2.5 x 10(4) copies/ml HBV DNA levels were optimal for discriminating CHB patients (HBeAg-negative) from inactive carriers with 75.6 and 78.6% sensitivity and specificity, respectively. Despite the extensive overlapping of HBV DNA levels in inactive carriers and HBeAg negative CHB patients, 2.5 x 10(4) copies/mL is the most favourable cut-off value to classify these individuals and would be imperative in the better management of this dreadful disease.

Multiplex Quantitative Competitive Polymerase Chain Reaction Based on a Multianalyte Hybridization Assay Performed on Spectrally Encoded Microspheres

Quantitative polymerase chain reaction (PCR) may be performed by two general approaches, namely, real-time PCR and quantitative competitive PCR (QC-PCR). QC-PCR makes use of the concept of a DNA competitor, which is the "gold standard" approach to circumvent the problem of the variation of amplification efficiency. However, QC-PCR in its classical form is a low-throughput method since it requires titration of each sample with the competitor followed by electrophoresis. The throughput of QC-PCR has been improved by capillary electrophoresis and microtiter well-based hybridization assays. The present work introduces a multiplex QC-PCR method, which is based on a multianalyte hybridization assay that is performed on spectrally encoded microspheres. The DNA competitors use the same primers and have equal size with their corresponding target DNA sequences but differ in a short (24 bp) centrally located sequence. Following multiplex PCR, biotinylated amplification products from all DNA targets and competitors are heat-denatured and hybridized with oligonucleotide probes, which are attached to addressable sets of fluorescent microspheres. The hybrids react with a streptavidin-phycoerythrin conjugate. The microspheres are then analyzed by flow cytometry employing two lasers. A red laser line is used for classification of the microspheres, and a green line excites phycoerythrin, whose fluorescence is related to the concentration of the analyte DNA. As a model, we have developed a multiplex quantitative competitive PCR assay for four targets. The amplification products from targets and competitors (a total of 8 DNA fragments) are determined simultaneously by the multianalyte hybridization assay. The limits of quantification for the hybridization assay of all amplified DNA fragments are below 13 pM. The multiplex quantitative competitive PCR assay detects approximately 500 copies from each target DNA. To our knowledge, the proposed method is the only approach to quantitative PCR that offers such a high potential for multiplexing.

A Simple, Rapid, and Sensitive Method for the Detection of theJAK2V617F Mutation

The point mutation 1849 (GT) V617F in the JAK2 gene occurs at high frequency in several chronic myeloproliferative diseases. Although a number of V617F mutation detection methods have been described, few are readily implemented in a diagnostic setting. We developed a simple and sensitive allelespecific competitive blocker polymerase chain reaction (ACB-PCR) assay to detect the V617F mutation. DNA was extracted from peripheral whole blood samples of 26 patients with chronic myeloproliferative disease. The ACB-PCR limit of detection was 1%. All positive samples detected by sequencing were detected by ACB-PCR. In 3 patients with essential thrombocythemia, the V617F mutation was readily detected by ACB-PCR but was near the detection limit of sequencing, confirming that ACB-PCR is more effective at detecting V617F when the mutant cell population is low. Detection of the monomorphic JAK2 V617F mutation using the ACB-PCR assay is easy to perform, rapid, sensitive, and cost-effective, which are key features of an ideal diagnostic method.

Quantification of the biocontrol agent Pseudomonas fluorescens Pf153 in soil using a quantitative competitive PCR assay unaffected by variability in cell lysis- and DNA-extraction efficiency

Although often neglected, variability in cell lysis efficiency and DNA extraction yield represents the major hurdles of any polymerase chain reaction (PCR)-based quantification protocol in soil and other natural environments. In this study we developed a technique that minimizes the effects of these constraints, providing at the same time a reliable internal control to distinguish between PCR-inhibition and negative results. We used Pseudomonas fluorescens Pf153, a root-colonizing bacterium that shows biocontrol activity against tobacco and cucumber black root rot, as the target organism for PCR quantification. Prior to DNA extraction, the genetically engineered, cognate reference strain P. fluorescens CHA0/c2 was inoculated in a reference soil. CHA0/c2 in the reference soil and Pf153 in the soil sample were lysed in parallel and afterward the lysates were mixed in known proportions. CHA0/c2 carries the plasmid pME6031-cmp2 that contains an allelic variant (competitor) of the Pf153 specific sequence Pf153_2. In a quantitative competitive PCR (QC-PCR) assay the competitor allows the quantification of the target strain down to 0.66 Pf153 CFU/mg soil. Processing the reference strain in the same way as Pf153 enables the exact quantification of the target strain in biocontrol assays performed in natural soil, overcoming differences in DNA extraction efficiency and PCR amplification from different soil environments. This technique is easily adaptable to other Pseudomonas strains simply by replacing the competitor used here with one derived from a SCAR-marker which is specific for the strain of choice.