Green qPCR Assay Research Articles

Development of a loop-mediated isothermal amplification assay for the detection and quantification of epizootic epitheliotropic disease virus (salmonid herpesvirus-3)

Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus-3) causes a serious disease hatchery-reared lake trout (Salvelinus namaycush), threatening restoration efforts of this species in North America. The current inability to replicate EEDV in vitro necessitates the search for a reproducible, sensitive, and specific assay that allows for its detection and quantitation in a time- and cost-effective manner. Herein, we describe a loop-mediated isothermal amplification (LAMP) assay that was developed for the quantitative detection of EEDV in infected fish tissues. The newly developed LAMP reaction was optimized in the presence of calcein, and the best results were produced using 2 mM MgCl2, 1.8 mM dNTPs and at an incubation temperature of 67.1 °C. This method was highly specific to EEDV, as it showed no cross-reactivity with several fish viruses, including Salmonid Herpesvirus-1, -2, -4, and -5, Infectious Pancreatic Necrosis Virus, Spring Viremia of Carp Virus, Infectious Hematopoietic Necrosis Virus, Golden Shiner Reovirus, Fathead Minnow Nidovirus, and Viral Hemorrhagic Septicemia Virus. The analytical sensitivity of the EEDV-LAMP method was estimated to be as low as 16 copies of plasmid per reaction. When infected fish tissue was used, a positive reaction could be obtained when an infected gill tissue sample that contained 430 viral copies/μg was diluted up to five orders of magnitude. The sensitivity and specificity of the newly developed LAMP assay compared to the SYBR Green qPCR assay were 84.3% and 93.3%, respectively. The quantitative LAMP for EEDV had a correlation coefficient (R2 = 0.980), and did not differ significantly from the SYBR Green quantitative PCR assay (p > 0.05). Given its cost- and time-effectiveness, this quantitative LAMP assay is suitable for screening lake trout populations and for the initial diagnosis of clinical cases.

Two mutations in the truncated Rep gene RBR domain delayed the Wheat dwarf virus infection in transgenic barley plants

Wheat dwarf virus (WDV), an important cereal pathogen, is closely related to Maize streak virus (MSV), a model virus of the Mastrevirus genus. Based on its similarity to known MSV resistance strategies, a truncated part of the WDV replication-associated (RepA) gene (WDVRepA215) and the WDV RepA gene with a mutated retinoblastoma-related protein (RBR) interaction domain (WDVRepA215RBRmut) were cloned into the pIPKb002 expression vector and transformed into immature embryos of spring barley cv. Golden Promise plants through Agrobacterium-mediated transformation. A detailed study of T1-generation plants infected by leafhoppers (Psammotettix alienus) fed on infection sources of variable strength was performed over a 5-week period encompassing the initial stages of virus infection. A DNA WDV TaqMan qPCR assay normalized using the DNA puroindoline-b SYBR Green qPCR assay for samples on a per week basis revealed an approximately 2-week delay in WDVRepA215RBRmut plants to WDVRepA215 plants before significant increases in the WDV viral levels occurred. Both WDVRepA215 and WDVRepA215RBRmut plants showed similar levels of transgenic transcripts over the screened period; however, the transgenic plants also showed increased numbers of infected plants compared to the control plants.

Identification of the Major Diacylglycerol Acyltransferase mRNA in Eukaryotic Cells

Triacylglycerols (TAGs) are the primary form of energy storage in eukaryotes. They also serve as a reservoir of fatty acids for membrane biogenesis of the cells and lead to obesity when excessively accumulated in adipose tissues. Diacylglycerol acyltransferases (DGATs) are responsible for the final and rate-limiting step of TAG biosynthesis in eukaryotic organisms. Understanding the roles of DGATs will help to create transgenic plants and microbes with value-added properties and provide information for therapeutic intervention for obesity and related diseases. Mammalian DGATs are divided into DGAT1 and DGAT2 subfamilies. It was unclear which DGAT was the major isoform expressed in animals. The objective of this study was to identify the major form of DGATs expressed in animal cells and compared to those expressed in tung tree seeds. TaqMan and SYBR Green qPCR assays evaluated DGAT mRNA levels in mouse 3T3-L1 adipocytes and RAW264.7 macrophages and tung tree seeds. TaqMan qPCR showed that DGAT mRNA levels were stable during the time-course study, DGAT2 mRNA levels were 10–30 folds higher than DGAT1 in adipocytes and macrophages, and DGAT mRNA levels in adipocytes were 50–100 folds higher than those in macrophages. As a positive control, the anti-inflammatory tristetraprolin (TTP) mRNA levels were 2–4 folds higher in macrophages than those in adipocytes and similar to DGAT1 in adipocytes but 100-fold higher than DGAT1 in macrophages. SYBR Green qPCR analyses confirmed TaqMan qPCR results. DGAT2 mRNA as the major DGAT mRNA in mouse cells was similar to that in tung tree seeds where DGAT2 mRNA levels were 10–20-fold higher than DGAT1 or DGAT3. Our results conclusively demonstrated that DGAT2 mRNA was the major form of DGATs expressed in plant and animal cells. This finding should facilitate efforts to regulate lipid/oil accumulation in plants and animals. Support or Funding Information Supported by USDA-ARS Quality and Utilization of Agricultural Products Research Program 306 through CRIS 6054-41000-103-00D. DGAT2 mRNA as the major form in eukaryote cells This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Detection and discrimination of five E. coli pathotypes using a combinatory SYBR\xae Green qPCR screening system

A detection and discrimination system for five Escherichia coli pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic E. coli (CoSYPS Path E. coli). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of E. coli: shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) E. coli. The SYBR® Green qPCR assays target the uidA, ipaH, eae, aggR, aaiC, stx1, and stx2 genes. uidA controls for E. coli presence and all the other genes are specific targets of E. coli pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path E. coli system was subsequently evaluated on four food matrices artificially contaminated with pathogenic E. coli. It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.

Developing a Real-Time PCR Assay for Direct Identification and Quantification of Pratylenchus penetrans in Soil.

The root-lesion nematode Pratylenchus penetrans is a major pathogen of potato worldwide. Yield losses may be exacerbated by interaction with the fungus Verticillium dahliae in the potato early dying disease complex. Accurate identification and quantification of P. penetrans prior to planting are essential for developing effective integrated pest control measures. However, distinction between P. penetrans and other Pratylenchus spp. based on morphology is a tedious task. A SYBR Green I-based qPCR assay was developed to discriminate, identify, and quantify P. penetrans in field soil. P. penetrans-specific qPCR primers were designed from the D2-D3 region of the 28S rDNA. The specificity of the assay was evaluated using eight isolates of P. penetrans populations and 31 isolates of other nematode species. A standard curve relating threshold cycle and log values of nematode number was generated from artificially infested soils. There was a high correlation between the P. penetrans numbers artificially added to soil or estimated from naturally infested field soils by conventional methods, and the numbers quantified using the qPCR assay. Grinding the field soil prior to DNA extraction improved P. penetrans detection from soil. The qPCR assay will not only be useful for differentiating P. penetrans from mixed populations of Pratylenchus spp., but also for efficient detection and quantification of P. penetrans from field soil. The assay requires no expertise in nematode taxonomy and morphology, and may serve as a useful diagnostic tool in research, diagnostic labs, and extension services for pest management.

Quantitative SYBR Green qPCR technique for the detection of the nematode parasite Anisakis in commercial fish-derived food

The extensive presence of anisakids in fish for human consumption has become a problem of food safety and quality. The aim of this study was to develop and assess the performance of a quantitative SYBR Green qPCR assay for the detection and quantification of Anisakis DNA in fish by-products. L3 nematode larvae of A. simplex (s.l.) (n=510), A. physeteris (n=3), Hysterothylacium sp. (n=10) and Pseudoterranova sp. (n=1), isolated from blue whiting, horse mackerel and monkfish, were used for the optimization of the molecular assay. In addition, molecularly typed larvae of A. simplex (s.s.) (n=10) and A. pegreffii (n=5) of the complex A. simplex (s.l.) were used for the specificity assay. Primers targeting the mitochondrial cytochrome c oxidase subunit II gene (COII) were selected. Analytical sensitivity and reproducibility were evaluated in a food matrix consisting of commercial fish-derived food spiked with larvae of A. simplex (s.l.). The assay proved to be specific for the three analyzed Anisakis species. A high reproducibility and sensitivity was detected, with a 95% limit of detection (LOD) of 0.30ng (95%CI 0.15–1.50) of A. simplex (s.l.) DNA per gram of food matrix and an operative LOD of 1.50ng after a PROBIT analysis. The assay was applied to study the presence of Anisakis in four types of processed commercial food, namely crab sticks, “gulas”, croquettes and burgers. Overall, 180 food samples from 15 commercial brands were studied, detecting Anisakis DNA in over half of them. The analyzed surimi-based products, “gulas” and crab sticks, showed the highest Anisakis burden (5.86±0.69 and 4.68±0.73ng of Anisakis DNA per gram of food, respectively). Our results indicate that the optimized SYBR Green qPCR technique is an accurate and sensitive method that may improve detection of Anisakis in fresh and processed products.

Comparing qPCR and Nested PCR Diagnostic Methods for Aster Yellows Phytoplasma in Aster Leafhoppers.

The aster yellows phytoplasma (AYp) is a wall-less bacterium that causes damage in multiple crops. They are spread primarily by the aster leafhopper, Macrosteles quadrilineatus (Hemiptera: Cicadellidae). A total of 3,156 aster leafhoppers were collected during the 2014 and 2015 growing seasons in Michigan celery and carrot fields using sweep nets. The objective of this study was to test previously developed 16S rDNA phytoplasma gene primers to find the most reliable and least time-consuming method for AYp detection in leafhoppers. Nested polymerase chain reaction (PCR) was performed with universal primers P1/P7 and R16F2n/R16R2, and then, restriction enzymes AluI, MseI, and HhaI identified the phytoplasma to subgroup. Over the two years, 2.2% of samples were phytoplasma positive with nested PCR, classified in subgroups 16SrI-A or 16SrI-B. All samples were also tested with a TaqMan quantitative qPCR assay with universal phytoplasma primers and probe and 4.6% tested positive. A subset of samples were also tested with AYp-specific SYBR green qPCR, showing a >93% similarity between SYBR green and TaqMan qPCR assay results. The qPCR assays were more than two times faster than nested PCR. However, qPCR assays likely have specificity issues that need to be addressed before they can be used as a reliable method of detection for AYp in leafhoppers.

Development of a quantitative PCR for the detection of Rangelia vitalii

The aim of this study was to develop and validate a SYBR® Green qPCR assay to detect and quantify a fragment of the 18S rRNA gene of Rangelia vitalii in canine blood. Repeatability of the qPCR was determined by the intra- and inter-assay variations. The qPCR showed efficiency of E=101.30 (r2=0.996), detecting as few as one copy of plasmid containing the target DNA. Specificity of the assay was performed using DNA samples of Babesia canis, B. gibsoni, Ehrlichia canis, E. ewingii and Leishmania sp. No cross-reactivity was observed. Field samples consisting of blood from 265 dogs from Porto Alegre, Brazil were also tested. A total of 24 (9.05%) samples were positive for R. vitalii. Amplicons of 50% of positive samples were confirmed to be R. vitalii by Sanger sequencing. The positive samples had an average of 3.5×105 organisms/mL of blood (range: 1.27×103–1.88×106) based on the plasmid-generated standard curve. In conclusion, the SYBR® Green qPCR assay developed herein is sensitive and specific and can be used as a diagnostic tool for detection and quantification of R. vitalii in canine blood samples.

QUANTITATIVE VS. CONVENTIONAL PCR FOR DETECTION OF HUMAN ADENOVIRUSES IN WATER AND SEDIMENT SAMPLES.

SUMMARYHuman Adenoviruses (HAdV) are notably resistant in the environment. These agents may serve as effective indicators of fecal contamination, and may act as causative agents of a number of different diseases in human beings. Conventional polymerase chain reaction (PCR) and, more recently, quantitative PCR (qPCR) are widely used for detection of viral agents in environmental matrices. In the present study PCR and SYBR(r)Green qPCR assays were compared for detection of HAdV in water (55) and sediments (20) samples of spring and artesian wells, ponds and streams, collected from dairy farms. By the quantitative methodology HAdV were detected in 87.3% of the water samples and 80% of the sediments, while by the conventional PCR 47.3% and 35% were detected in water samples and sediments, respectively.

Highly Sensitive End-Point PCR and SYBR Green qPCR Detection of Phymatotrichopsis omnivora, Causal Fungus of Cotton Root Rot.

Phymatotrichopsis omnivora, the causal pathogen of cotton root rot, is a devastating ascomycete that affects numerous important dicotyledonous plants grown in the southwestern United States and northern Mexico. P. omnivora is notoriously difficult to isolate from infected plants; therefore methods for accurate and sensitive detection directly from symptomatic and asymptomatic plant samples are needed for disease diagnostics and pathogen identification. Primers were designed for P. omnivora based on consensus sequences of the nuclear ribosomal internal transcribed spacer (ITS) region of geographically representative isolates. Primers were compared against published P. omnivora sequences and validated against DNA from P. omnivora isolates and infected plant samples. The primer combinations amplified products from a range of P. omnivora isolates representative of known ITS haplotypes using standard end-point polymerase chain reaction (PCR) methodology. The assays detected P. omnivora from infected root samples of cotton (Gossypium hirsutum) and alfalfa (Medicago sativa). Healthy plants and other relevant root pathogens did not produce PCR products with the P. omnivora-specific primers. Primer pair PO2F/PO2R was the most sensitive in end-point PCR assays and is recommended for use for pathogen identification from mycelial tissue and infected plant materials when quantitative PCR (qPCR) is not available. Primer pair PO3F/PO2R was highly sensitive (1 fg) when used in SYBR Green qPCR assays and is recommended for screening of plant materials potentially infected by P. omnivora or samples with suboptimal DNA quality. The described PCR-based detection methods will be useful for rapid and sensitive screening of infected plants in diagnostic laboratories, plant health inspections, and plant breeding programs.

Comparison of two poultry litter qPCR assays targeting the 16S rRNA gene of Brevibacterium sp.

Chicken feces commonly contain human pathogens and are also important sources of fecal pollution in environmental waters. Consequently, methods that can detect chicken fecal pollution are needed in public health and environmental monitoring studies. In this study, we compared a previously developed SYBR green qPCR assay (LA35) to a novel TaqMan qPCR assay (CL) for the environmental detection of poultry-associated fecal pollution. We tested both assays against chicken litter (n = 40), chicken fecal samples (n = 186), non-chicken fecal sources (n = 484), and environmental water samples (n = 323). Most chicken litter samples (i.e., ≥98%) were positive for both assays with relatively high signal intensities, whereas only 23% and 12% of poultry fecal samples (n = 186) were positive with the LA35 and the CL assays, respectively. Data using fecal samples from non-target animal species showed that the assays are highly host-associated (≥95%). Bayesian statistical models showed that the two assays are associated with relatively low probability of false-positive and false-negative signals in water samples. The CL marker had a lower prevalence than the LA35 assay when tested against environmental water samples (i.e., 21% vs. 31% positive signals). However, by combining the results from the two assays the detection levels increased to 41%, suggesting that using multiple assays can improve the detection of chicken-fecal pollution in environmental waters.

SYBR®Green qPCR Salmonella detection system allowing discrimination at the genus, species and subspecies levels

In this work, a three-level Salmonella detection system based on a combination of seven SYBR®Green qPCR was developed. This detection system discriminates Salmonella at the genus, species and subspecies levels using a single 96-well plate. The SYBR®Green qPCR assays target the invA, rpoD, iroB and safC genes, as well as the STM0296 locus, putatively coding for a cytoplasmic protein. This study includes the design of primer pairs, in silico and in situ selectivity, sensitivity, repeatability and reproducibility evaluations of the seven SYBR®Green qPCR assays. Each detection level displayed a selectivity of 100 %. This combinatory SYBR®Green qPCR system was also compared with three commercially available Salmonella qPCR detection kits. This comparison highlighted the importance of using a multi-gene detection system to be able to detect every target strain, even those with deletion or mutation of important genes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-013-5234-x) contains supplementary material, which is available to authorized users.

Spinal muscular atrophy carrier frequency in Ukraine

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder with the frequency of carriers in a number of ethnical groups ranging from 1/50 to 1/25. However, the prevalence of SMA for population of Ukraine remains to be established. For the analysis of deletion in exon 7 SMN1 gene in SYBR Green Real-Time qPCR assay specific for the single nucleotide change in exon 7 (cd 840 C >T) was used. Using SYBR Green qPCR assay, the incidence of the exon 7 SMN1 deletion was established among 370 unrelated individuals without family history of SMA. The carrier frequency for this group of Ukrainians was estimated as 3.24% (1/31). The results of our study showing the high prevalence of carriers warrant the importance of population screening for SMA in Ukraine.

Characterization of reference gene expression in tung tree (Vernicia fordii)

Tung oil from tung tree (Vernicia fordii) is widely used as a drying ingredient in paints, varnishes, and other coatings and finishes. Recent research has focused on the understanding of the biosynthesis of oil in tung trees. Many oil biosynthetic genes have been identified in tung tree but little is known about the expression patterns of the genes in tung seeds. Quantitative real-time-PCR (qPCR) assays are widely used for gene expression analysis. One crucial task of qPCR assay design is to select stably expressed internal reference genes for data analysis. The objective of this study was to characterize the expression of potential reference genes in the tung tree to provide a sound basis for reliable and reproducible qPCR results. The expression of tung 60s ribosome protein L19 (Rpl19b), glyceraldehyde 3 phosphate dehydrogenase (Gapdh) and ubiquitin protein ligase (Ubl) was examined by TaqMan and SYBR Green qPCR using RNA from three trees, three tissues (seeds, leaves and flowers) and 11 time events of developing seeds. The variations of the three mRNA levels were compared between two RNA extraction methods, two cDNA preparations, and the same or different PCR plates. Overall results demonstrated that Rpl19b was the most stably expressed gene, followed closely by Ubl, and Gapdh was the worst among the three genes under optimized qPCR assay conditions. These results suggest that Rpl19b and Ubl are preferable reference genes for both TaqMan and SYBR Green qPCR assays. The development of these reference genes for quantitative gene expression analyses in tung trees should facilitate identifying target genes for genetic engineering industrial oils in oilseed crops.

Abstract LB-68: Identification of circulating HPV DNA in urine with a dual sequence capture approach.

Abstract HPV testing is increasingly used for cervical cancer screening in conjunction with cervical cytology. Privacy, cultural, and infrastructure issues challenge the effective implementation of HPV testing in cervical cancer screening. The detection of circulating HPV DNA from urine can be useful for cervical cancer screening. We captured and sequenced the HPV genome after isolating HPV Transrenal DNA (HPV-TrDNA) and then developed a qPCR-based HPV-TrDNA assay to quantify high risk HPV TrDNA. HPV TrDNA molecules are relatively short, fragmented, pieces of circulating DNA (less than 250 nucleotides) that get filtered by the kidneys and can be isolated from urine. Methods: We adsorbed cell-free nucelic acids from urine samples (10mL) of 41 women with 23 LGL and 18 HGL cervical lesions and 31 women with normal cytology on a Q-Sepharose resin followed by a silica-based method. A custom-designed pool of HPV-specific dual sequence capture probes was used for library amplification and target selection (Roche/NimbleGen SeqCap EZ Choice Library). A pre-capture amplification of the library was performed with ligation-mediated PCR (LM-PCR) using primers complementary to the adaptors, followed by two rounds of hybridization to the HPV-specific SeqCap EZ Choice Library. Amplified captured DNA from HPV infected cell lines, HeLa (HPV 18), CSCC7 (HPV16), and HPV TrDNA samples from premalignant cervical lesions were processed for multiplexed sequencing on the GS Junior system (Roche). Following completion of the pyrosequencing run, signal processing was performed, followed by detailed analyses. For the SYBR green qPCR assay we amplified an HPV E1 region common to thirteen high-risk HPV types for cervical cancer. Results: We isolated TrDNA from 10mL of urine samples. The mean concentration, total amount, and 260/280 ratio of isolated TrDNA was 34.2 ng/ul;1ug and 1.7 for premalignant samples and 17.2 ng/ul; 0.5ug; and 1.4 for normal samples. The total number of Pass Filter sequence reads was 100,428, the total number of bases over 41 million and the read length average was 130.3 bp. Most reads (91.63%) mapped to the reference HPV sequences, with the total number of mapped base pairs over 36 million (88%). The number of fully mapped reads was 14,899 (14.84%), and the number of partially mapped reads was 70,686 (88.17%). The average coverage of reference sequences was 78% for HPV 18 in the CSCC7 DNA, 86% for HPV 16 in HeLa DNA, as well as, 100% for HPV 18 and 96% for HPV 16 in TrDNA samples. The average Ct value for Pre-Capture LM-PCR DNAs was 35.74. The average Ct value for Post-Capture LM-PCR DNAs was 19.73; therefore the Delta Ct value was 16.01. Based on an estimated efficiency for the assay, the approximate fold enrichment was greater than 12,000. Conclusions: We demonstrated that the HPV genome can be assembled from HPV TrDNA. We are also the first to show that circulating high risk HPV TrDNA can be quantified using SYBR green qPCR assay after dual sequence capture of the HPV genome. Citation Format: Rafael E. Guerrero-Preston, Anne Jedlicka, Teresa Diaz-Montes, Josefina Romaguera, Juan C. Roa, David Sidransky. Identification of circulating HPV DNA in urine with a dual sequence capture approach. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-68. doi:10.1158/1538-7445.AM2013-LB-68

Comparison of TaqMan and SYBR Green qPCR Methods for Quantitative Gene Expression in Animals

Quantitative real‐time‐PCR (qPCR) such as TaqMan and SYBR Green qPCR assays are widely used for gene expression analysis. The drawbacks of SYBR Green assays include that the dye is nonspecific which can generate false positive signals if non‐specific products or primer‐dimers are present in the assay and that the length of the amplicon affects the intensity of the amplification. However, little was reported in the literature on direct comparison of both methods using the same pairs of primers and biological samples. We therefore compared both assays using 24 RNAs from mouse 3T3‐L1 adipocytes, RAW264.7 macrophages, and rat adipocytes, brain, kidney, liver, and muscle tissues. High quality RNA with high rRNA ratio and RNA integrity number was isolated from these animal cells and tissues and used for cDNA synthesis and qPCR amplification. The surprising finding was that SYBR Green qPCR over‐estimated the expression levels by several folds in most of the genes and tissues tested. This was unlikely due to non‐specific PCR products or the length of PCR products. The results demonstrate that both assays are reliable for determining gene expression levels in cells and tissues, but that TaqMan assay generally generates lower calculated expression levels than SYBR Green assay. This study suggests that any discussion of gene expression levels requires specification of qPCR methods used in the analysis.

Development and validation of qualitative SYBR®Green real-time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes.

A combination of four qualitative SYBR®Green qPCR screening assays targeting two levels of discrimination: Listeria genus (except Listeria grayi) and Listeria monocytogenes, is presented. These assays have been developed to be run simultaneously using the same polymerase chain reaction (PCR) programme. The paper also proposes a new validation procedure to specifically validate qPCR assays applied to food microbiology according to two guidelines: the ISO 22118 norm and the “Definition of minimum performance requirements for analytical methods of GMO testing”. The developed assays target the iap, prs and hlyA genes that belong to or neighbour the virulence cluster of Listeria spp. The selected primers were designed to amplify short fragments (60 to 103 bp) in order to obtain optimal PCR efficiency (between 97 and 107 % efficiency). The limit of detection of the SYBR®Green qPCR assays is two to five copies of target genes per qPCR reaction. These assays are highly accurate (98.08 and 100 % accuracy for the Listeria spp. and L. monocytogenes assays, respectively).

Towards the establishment of a consensus real-time qPCR to monitor Trypanosoma cruzi parasitemia in patients with chronic Chagas disease cardiomyopathy: A substudy from the BENEFIT trial

Quantitative real-time PCR (qPCR) is an accurate method to quantify Trypanosoma cruzi DNA and can be used to follow-up parasitemia in Chagas disease (CD) patients undergoing chemotherapy. The Benznidazole Evaluation for Interrupting Trypanosomiasis (BENEFIT) study is an international, multicenter, randomized, double-blinded and placebo-controlled clinical trial to evaluate the efficacy of benznidazole (BZ) treatment in patients with chronic Chagas cardiomyopathy (CCC). One important question to be addressed concerns the effectiveness of BZ in reducing overall parasite load in CCC patients, even in the absence of parasitological cure. This report describes the evaluation of multiple procedures for DNA extraction and qPCR-based protocols aiming to establish a standardized methodology for the absolute quantification of T. cruzi DNA in Guanidine-EDTA blood (GEB) samples. A panel of five primer sets directed to the T. cruzi nuclear satellite DNA repeats (Sat-DNA) and to the minicircle DNA conserved regions (kDNA) was compared in either SYBR Green or TaqMan systems. Standard curve parameters such as, amplification efficiency, coefficient of determination and intercept were evaluated, as well as different procedures to generate standard samples containing pre-established T. cruzi DNA concentration. Initially, each primer set was assayed in a SYBR Green qPCR to estimate parasite load in GEB samples from chronic Chagas disease patients. The results achieved from Bayesian transmutability analysis elected the primer sets Cruzi1/Cruzi2 (p=0.0031) and Diaz7/Diaz8 (p=0.0023) coupled to the QIAamp DNA Kit extraction protocol (silica gel column), as the most suitable for monitoring parasitemia in these patients. Comparison between the parasite burden of 150 GEB samples of BENEFIT patients from Argentina, Brazil and Colombia, prior to drug/placebo administration, was performed using Cruzi1/Cruzi2 primers in a SYBR Green approach. The median parasitemia found in patients from Argentina and Colombia (1.93 and 2.31 parasite equivalents/mL, respectively) was around 20 times higher than the one estimated for the Brazilian patients (0.1 parasite equivalents/mL). This difference could be in part due to the complexity of T. cruzi genetic diversity, which is a factor possibly implicated in different clinical presentations of the disease and/or influencing parasitemia levels in infected individuals from different regions of Latin America. The results of SYBR Green qPCR assays herein presented prove this methodology to be more cost efficient than the alternative use of internal fluorogenic probes. In addition, its sensitivity and reproducibility are shown to be adequate to detect low parasitemia burden in patients with chronic Chagas disease.

18-OR

Aim Natural Killer cells cause elimination of malignant and virus-infected cells by their cell surface receptors namely Killer-cell Immunoglobulin-like Receptors (KIR). Current KIR genotyping methods use sequence-based typing or sequence-specific primer or oligonucleotide probe (SSP/SSO) based PCR followed by agarose gel electrophoresis. Our aim was to evaluate the feasibility of using a quantitative PCR (qPCR) based research assay for KIR genotyping with minimal DNA input and reduced turn around time with potential for a multiplex KIR genotyping assay in a single tube. Methods A panel of 24 DNA containing common KIR genotypes was obtained from UCLA. 85 ng total DNA for 16 KIR reactions was used for the qPCR research assays compared to 2-3 μg DNA in a SSP-PCR commercial kit. DNA genotypes were confirmed with a SSP-PCR commercial kit and results were compared using the developed qPCR research assay. 32 primers and 16 FAM labeled probes were designed for this assay. The same primer set was also validated using a SYBR Green qPCR assay. A qPCR internal positive control accounts for true absence of KIR alleles. Results KIR research qPCR and SYBR Green qPCR assay genotyping results were 100% concordant with the commercial SSP-PCR kit. Our developed assay could identify the presence or absence of each KIR allele with a 35× reduction in DNA input with no post-PCR analyses or additional specialized equipment. Conclusions We have developed, optimized and validated a qPCR research assay for KIR genotyping that is robust and flexible for both small and large scale genetic analysis studies with potential for high throughput protocols. With more associations of KIR and disease outcomes in autoimmunity, infectious diseases, pregnancy-related complications and in transplantation, a more efficient and sensitive qPCR assay with faster detection might aid earlier therapy intervention making genotyping KIRs using qPCR based assays ideal in clinical and in basic research settings. Wadia: Quest Diagnostics: Employee; Stockholder. Bost: Quest Diagnostics: Employee.

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.