Primer Dimers Research Articles

The Use of Immunoglobulin Gene Rearrangement Polymerase Chain Reaction Assays for Detection of B-Cell Clonality for Plasma Cell Neoplasms Using Novel PCR Primers

We have designed a standardized protocol with multiplex-primer sets capable of detecting majority IG kappa (IGK) and IG lambda (IGL) light chain rearrangements in plasma cell neoplasms (PCN). Thirty primers were combined in three multiplexed PCR reactions to target IGK, KDE and IGL rearrangements. Variable region (V) primers were designed to prevent “primer dimers”, provide matching melting temperatures (Tm), minimize amplicon size, and optimize sequencing time. Amplicons were subjected to capillary gel electrophoresis for analysis. In a discovery series, we tested 37 plasma cells neoplasms PCN (28 PCNs at diagnosis and 9 PCNs post-treatment). The assay investigated an additional 52 prospective PCN cases in the validation series. Results were compared to bone marrow morphology, immunohistochemical (IHC), flow cytometry data, and standard IGH FRIII gene rearrangement assay. In the discovery series, the following sensitivities/specificities were obtained for mature B-cell neoplasms: IGH FRIII: 29.7%/100%, IGK: 80.4%/100%, KDE: 25.0%/100%, and IGL: 35.1%/96.8%. The combination of IGH FRIII, IGK, and KDE detected 83.8% (31/37) vs 67.3% (35/52) in the discovery vs validation series, respectively, for the PCN population. Interestingly, 21.2% (11/52) of the validation samples positive by IG clonality, were negative by IHC and flow cytometry. In IHC/flow cytometry positive cases with a PCN representing a tumor burden of >50%, 10% to 50%, 1% to 10%, 0% to 1% of cells, the combined sensitivity of the IG clonality assay was 100% (20/20), 72% (23/32), 53% (10/19) and 20% (1/5) respectively. This IGK/IGL clonality assay has good sensitivity at diagnosis.

Helicase-Dependent Isothermal Amplification of DNA and RNA by Using Self-Avoiding Molecular Recognition Systems.

Assays that detect DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low-resource environments, and requires equipment and power that might not be available in these environments. Isothermal procedures, which avoid thermal cycling, are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a "self avoiding molecular recognition system" (SAMRS) eliminates these artifacts and gives clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3'-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, difficult to achieve with HDA using standard primers. Thus, SAMRS-HDA is a more versatile approach than standard HDA, with a broader applicability for xNA-targeted diagnostics and research.

Optimization of Real Time PCR for Precise Measurement of HER2 Overexpression in Breast Cancer Specimens

Background: Breast cancer is one of the most prevalent malignancies among women in various countries. HER2 overexpression, which is due to different reasons, occurs in 20-30% of breast cancers. HER2 gene encodes 185 kDa transmembrane glycoprotein with 1255 amino acids. This active product triggers downstream intracellular signaling pathways inducing cell proliferation and cell survival. These activities could be done in an uncontrolled manner when HER2 expression undergoes up-regulation. The aim of this study was optimization of Real Time PCR condition. Materials and Methods: RNA purification, cDNA synthesis and then optimization of Real Time PCR method performed respectively. In this study, total RNA was extracted from fresh tissue samples, first strand of total cDNA was synthesized and in the following steps, Real Time PCR was performed to be optimized. Results: Although altering the protocol, annealing temperature and concentration of MgCl2 did not make any improvement and beneficial effects on reactions, changing the concentration of primers to 0.24 pm/μl was influential to eliminate primer dimers of Real Time PCR reactions. It demonstrated that the copy number of GAPDH transcripts is more than HER2 transcripts in normal breast tissues. Therefore, deviation in 2.5 differences between the Ct value of HER2 and GAPDH indicated that the copy number of HER2 transcripts was increased; therefore, HER2 underwent overexpression in these cases. Conclusion: Under these optimized conditions, this technique can be applied as a powerful method in clinical laboratories.

Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification.

The technique of loop-mediated isothermal amplification (LAMP) utilizes four (or six) primers targeting six (or eight) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an increased likelihood of non-specific amplification induced by primer dimers. In this study, a set of LAMP primers were designed targeting the prfA gene sequence of Listeria monocytogenes, and dimethyl sulfoxide (DMSO) as well as Touchdown LAMP were employed to increase the sensitivity and specificity of the LAMP reactions. The results indicate that the detection limit of this novel LAMP assay with the newly designed primers and additives was 10 fg per reaction, which is ten-fold more sensitive than a commercial Isothermal Amplification Kit and hundred-fold more sensitive than previously reported LAMP assays. This highly sensitive LAMP assay has been shown to detect 11 strains of Listeria monocytogenes, and does not detect other Listeria species (including Listeria innocua and Listeria invanovii), providing some advantages in specificity over commercial Isothermal Amplification Kits and previously reported LAMP assay.

Role of the No Template Control samples in the selection of primers to quantify ‘Candidatusphytoplasma pyri’ by qPCR assays

Recent reports indicate as essential information, that must be submitted with the manuscripts about quantitative PCR the quantification cycle (Cq), corresponding to threshold cycle (Ct), of the samples named no template control (NTC), containing the mix without DNA template. Moreover if all samples without templateshow different Cq valuesfor at least threetimes, thereisahigh probability of contamination due to human error during the plate loading. In this case, samples that resulted positive in analytical PCR can differ only of one Ct from the NTC samples. This problem was found in checking two couples of primers to quantify ‘Candidatus Phytoplasma pyri’ in DNAs samples extracted from pear leaves that were positive in nested PCR assays. The possibility of a contamination of one of the reagents was excluded since the Ct values of NTC samples were not close to each other and the melting curve analysis did not reveal the presence of primer dimers at low temperatures. The analyses of NTC samples were determinant suggesting to not further promote the use of the two couples of primers.

PrimerDesign-M: a multiple-alignment based multiple-primer design tool for walking across variable genomes.

Analyses of entire viral genomes or mtDNA requires comprehensive design of many primers across their genomes. Furthermore, simultaneous optimization of several DNA primer design criteria may improve overall experimental efficiency and downstream bioinformatic processing. To achieve these goals, we developed PrimerDesign-M. It includes several options for multiple-primer design, allowing researchers to efficiently design walking primers that cover long DNA targets, such as entire HIV-1 genomes, and that optimizes primers simultaneously informed by genetic diversity in multiple alignments and experimental design constraints given by the user. PrimerDesign-M can also design primers that include DNA barcodes and minimize primer dimerization. PrimerDesign-M finds optimal primers for highly variable DNA targets and facilitates design flexibility by suggesting alternative designs to adapt to experimental conditions. PrimerDesign-M is available as a webtool at http://www.hiv.lanl.gov/content/sequence/PRIMER_DESIGN/primer_design.html tkl@lanl.gov or seq-info@lanl.gov.

Comparison of SYBR Green and TaqMan real-time PCR methods for quantitative detection of residual CHO host-cell DNA in biopharmaceuticals

The Chinese hamster ovary (CHO) cell line is one of the predominant hosts used in the bioproduction of pharmaceutical proteins. There have been many concerns about the use of animal cell lines in biopharm industries, and one of the most important concerns has been residual host-cell DNA. Improper integration of residual DNA into the recipient genomes could activate oncogenes or deactivate tumor suppressor genes. Real-time polymerase chain reaction (PCR) is a routine assay method used in the quantification of DNA. In this study, genomic CHO DNA was purified and subjected to real-time PCR. The efficiency of the reaction was calculated, and the limit of detection (LOD) was determined. The calculated efficiency for the primers using the SYBR Green method was 94.3% (r2 = 0.998). A melting curve analysis showed neither unspecific products nor primer dimers. The calculated efficiency for the TaqMan assay was 96.6% (r2 = 1). The results showed that the LOD of the SYBR Green and TaqMan assays were 100 fg and 10 fg, respectively. Since the LOD of the TaqMan assay showed a better sensitivity than the SYBR Green, this method could be used directly on the final products for the quantification of residual DNA, without prior DNA extraction.

Bovine Thrombin Enhances the Efficiency and Specificity of Polymerase Chain Reaction

The polymerase chain reaction (PCR) has become one of the central techniques in molecular biology since its invention. However, PCR can be fraught with difficulties in various situations, and it is desirable to find novel PCR enhancers suitable for universal applications. Here we show that bovine thrombin (BT), a well-known coagulation protein, is exceptionally effective at preventing the formation of primer dimers and enhancing the formation of the desired PCR products. The PCR enhancement effects of BT were demonstrated by testing various types of samples, including low-copy synthetic single-stranded DNAs (ssDNAs), synthetic ssDNA pools, human genomic DNA, and hepatitis B virus genomic DNA. In addition, BT was also able to effectively relieve PCR inhibition by nanomaterial inhibitors such as gold nanoparticles (AuNPs) and graphene oxide (GO). Compared with BSA, one of the most popular PCR enhancers, BT was more effective and required concentrations 18-178 times less than that of BSA to achieve a similar level of PCR enhancement.

Strand Invasion Based Amplification (SIBA®): a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2′-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

Abstract 4018: Development and analytical validation of a novel real-time multiplex RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR mRNA expression in circulating tumor cells of breast cancer patients

Abstract Introduction: Circulating Tumor Cells (CTCs) represent an important biological link in the spread of breast cancer from primary to metastatic disease. CTCs have already been established as strong predictors of prognosis in patients with metastatic breast cancer. Triple-negative breast cancers have a more aggressive clinical outcome than other forms of breast cancer. The aim of our work was to develop a quantitative real-time multiplex RT-qPCR assay for studying the expression of ER, PR, HER-2 and EGFR in CTC of breast cancer patients. Materials and Methods: A quadruplex quantitative real time RT-qPCR assay for ER, PR, HER-2 and EGFR was developed based on the de novo in-silico design of eight primers and four dual hybridization probes. All primers and probes were designed to avoid cross-reactions and primer dimer formation and to match the assay conditions, such as amplicon sizes, and melting temperatures. The specificity of all primer and hybridization probe sequences was first tested by homology searches in the nucleotide database (NCBI, nucleotide BLAST). Each probe set included a 3′-fluorescein (F) donor probe and a 5′- LC acceptor probe that was different for each gene set: ER at 610nm, PR at 640nm, HER-2 at 670nm, EGFR at 705nm. A color compensation test was performed by using pure dye spectra so that spectral overlap between dyes was corrected. The LightCycler 2.0 platform, (Roche, Diagnostics) was used since it allows detection of six multiple reporter dyes in the same capillary. The assay was designed, so that only 1 μL of cDNA is used and the total reaction volume is 10 μL. Results: The assay sensitivity was evaluated by performing spiking experiments. For this reason known numbers of MCF7 and SKBR-3 cells, as well as external quantification calibrators ranging from 105 copies/μL to 10 copies/μL, were used. The specificity was evaluated by carefully designed cross reaction studies, performed for each gene target in the presence of all other targets. Our assay, under optimized conditions has shown a very high specificity for each analyte. Conclusions: We report for the first time a highly sensitive, specific and reproducible quantitative multiplex real-time RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR. The assay is currently validated in CTC isolated from a large number of breast cancer patients. Citation Format: Areti D. Strati, Evi S. Lianidou. Development and analytical validation of a novel real-time multiplex RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR mRNA expression in circulating tumor cells of breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4018. doi:10.1158/1538-7445.AM2014-4018

Visual Detection of <i>Mycobacterium t</i><i>uberculosis</i> Complex with Loop-Mediated Isothermal Amplification and Eriochrome Black T

The present study was aimed to improve the method for detecting Mycobacterium tuberculosis complex recently reported on Letters in Applied Microbiology. Eriochrome black T (EBT) was added to the reaction system of LAMP, due to complexation of EBT with magnesium ions, the color of reaction solution was red at beginning, when the reaction was in process, the precipitate of magnesium ions and pyrophosphate ions formed, EBT lost magnesium ions, the color of reaction solution became sky blue from red gradually, and the method exhibits a high sensitivity of 8 fg/μL. The improved LAMP assay can detect Mycobacterium tuberculosis complex rapidly, sensitively and visually, the method developed in this study had solved the “bottleneck” in popularization and application of LAMP technology, which had not only avoided aerosol pollution but also solved the false positive problem caused by primer dimers.

1031-LBP: Evaluation of uracil dna glycosylase (UDG) with the LinkSeq™ real-time pcr hla typing method

Aim Post-PCR amplification products can pose a problem in clinical laboratories. Great care is needed to ensure post and pre-PCR areas minimize the risk of unwanted amplicons affecting results. Linkage Biosciences designed the LinkS e ¯ q™ real-time PCR HLA test to include deoxyuridine triphosphate (dUTP) rather than the standard deoxythmidine triphosphate (dTTP) in the PCR reaction as a preventive measure so that Uracil DNA Glycosylase enzyme (UDG) can be used as an optional additive to eliminate unwanted uracil-containing amplicons during PCR set-up. The aim of this study was to evaluate the effectiveness of adding UDG to the LinkS e ¯ q™ test in a laboratory setting with False Positive and False Negative issues. Methods To validate the usefulness of UDG reagent (provided by Linkage Biosciences) we selected 10 clinical DNA samples that contained aberrant primer dimer peaks which may have contributed to False Positive and False Negative allele amplification. All samples were typed with the standard LinkS e ¯ q™ protocol and with the UDG protocol. The 2 real-time kits that were used were the LinkS e ¯ q™ 384 well tray, Cat #1575 and the 192 well tray, Cat#1554 with SYBR Green dye for melting curve analysis. All samples were amplified using the Roche LightCycler® 480. Results All 10 samples showed improved results in the reduction of False Positives and removal of dimeric peaks using the UDG reagent. The data shows a total reduction of 93.6% of the False Positives, 66.6% reduction in False Negatives, and 86.7% reduction of software forced calls. The samples demonstrated concordance with PCR-SSP (Invitrogen), or UNET reported results. Conclusions In summary, the LinkS e ¯ q™ real-time PCR assay is a fast and reliable HLA typing method. The UDG reagent works well when necessary to remove suspect peaks which may interfere with SureTyper Software data analysis.

Establishment and evaluation of direct assay for plasma microRNAs without RNA extraction by quantitative PCR

Objective To establish a new method for the detection of plasma miRNAs by real-time fluorescence quantitative PCR without RNA extraction. Methods 7.5 μl plasma separated from normal human blood was treated with equal amount of buffer containing Tween 20, which was used directly as sample for the following reverse transcription and PCR reaction to establish a direct assay for plasma microRNAs. U6 snRNA, miR-24-1-5p and miR-4634 were selected as target microRNAs for the determination of RT-qPCR. Both specificity and amplification efficiency were assessed. The difference of Cq values between traditional quantitative PCR detection method and our new method was also observed. Furthermore, the results of mixed primer or individual primer by RT-qPCR were compared in new established method. Results The amplification products of U6, miR-24-1-5p and miR-4634 show a single melting peak at 81 ℃, 83 ℃ and 84.2 ℃, respectively, without primer dimer peak or non-specific peak in all 8 cases of healthy human plasma samples. Amplification efficiency for miR-24-1-5p reached 1.1 detected in plasma direct assay. The amplification curve of U6, miR-24-1-5p and miR-4634 is parallel in each sample, which demonstrated that the amplification of slope is consistent. The Cq value of miR-4634 determined by direct assay is higher than the result of the method by RNA extraction（plasma direct assay 30.98±0.17，RNA extraction 29.22±0.21；t=8.475，P 0.05）and miR-24-1-5p(plasma direct assay 20.73±0.14，RNA extraction 21.06±0.21；t=1.749, P>0.05) although they had significant correlation（r=0.860 5,0.728 9,0.748 5,P<0.05）. No significant difference of Cq value between mixed primer reverse transcription and individual primer reverse transcription. Conclusion A novel RT-qPCR direct assay of plasma miRNAs is established, which can improve the efficiency of plasma miRNAs determination in potential clinical applications. （Chin J Lab Med，2014,37：119-122） Key words: MicroRNAs; Real-time polymerase chain reaction; Plasma

Application Areas.

Application Areas.

A tool for design of primers for microRNA-specific quantitative RT-qPCR

BackgroundMicroRNAs are small but biologically important RNA molecules. Although different methods can be used for quantification of microRNAs, quantitative PCR is regarded as the reference that is used to validate other methods. Several commercial qPCR assays are available but they often come at a high price and the sequences of the primers are not disclosed. An alternative to commercial assays is to manually design primers but this work is tedious and, hence, not practical for the design of primers for a larger number of targets.ResultsI have developed the software miRprimer for automatic design of primers for the method miR-specific RT-qPCR, which is one of the best performing microRNA qPCR methods available. The algorithm is based on an implementation of the previously published rules for manual design of miR-specific primers with the additional feature of evaluating the propensity of formation of secondary structures and primer dimers. Testing of the primers showed that 76 out of 79 primers (96%) worked for quantification of microRNAs by miR-specific RT-qPCR of mammalian RNA samples. This success rate corresponds to the success rate of manual primer design. Furthermore, primers designed by this method have been distributed to several labs and used successfully in published studies.ConclusionsThe software miRprimer is an automatic and easy method for design of functional primers for miR-specific RT-qPCR. The application is available as stand-alone software that will work on the MS Windows platform and in a developer version written in the Ruby programming language.

Circulating microRNAs: Understanding the Limits for Quantitative Measurement by Real‐Time PCR

Ever since the discovery of small, non-coding RNAs in circulation, microRNAs have gained significant attention as biomarkers or, in some instances, regulators of disease and/or associated complications. Until now, most articles published use real-time quantitative PCR (qPCR) for assessment of

Getting Things Backwards to Prevent Primer Dimers

This Commentary highlights the article by Satterfield that describes a new class of primer technology-cooperative primers, which prevent primer-dimer amplification.

A Cross-Sectional Study of Helicobacter Infection Among Laboratory Animals and Animal Research Workers

A Cross-Sectional Study of Helicobacter Infection Among Laboratory Animals and Animal Research Workers

PCR em tempo real para detecção do vírus da doença de Aujeszky

O objetivo deste trabalho foi desenvolver uma PCR em tempo real (qPCR) para o diagnóstico rápido e sensível da doença de Aujeszky. Os iniciadores amplificaram um fragmento de 123 pares de base do gene codificante da glicoproteína D. A qPCR foi testada em 25 amostras de cérebro de suíno positivas e 85 amostras negativas para DA no isolamento viral e na soroneutralização. A sensibilidade analítica foi calculada com acréscimo de um isolado brasileiro do SuHV-1 titulado em amostras de cérebro de suíno negativas na soroneutralização e na PCR. A técnica apresentou sensibilidade analítica de 10-0,5 TCID50/50µL. A qPCR foi capaz de distinguir reações inespecíficas devido a dímero de oligonucleotídeos iniciadores ou amplificações, além do alvo designado (evitando, assim, os falso-positivos), e de obter resultados rápidos.

A Closed-Tube Detection of Loop-Mediated Isothermal Amplification (LAMP) Products Using a Wax-Sealed Fluorescent Intercalator

LAMP is an isothermal amplification method that can achieve ultra-high sensitivity and specificity. However, the conventional detection of LAMP amplicons can lead to cross-contamination due to the need to open the reaction tube which contains a large number of amplicons. To achieve closed-tube LAMP detection, we have developed a method that separates a solution of SYBR Green I (SGI) from the LAMP reagents using temperature-sensitive wax. The SGI is sealed in the bottom of the tube so not to interfere with the LAMP reaction, but is released into the mixture after the completion of the reaction by melting the wax. To enable the analysis of the closed-tube LAMP samples automatically, an instrument based on this new method was constructed. The background measurement of the LAMP due to primer dimers was significantly reduced by detecting the amplicons at 75 degrees C. HBV and 2009 H1N1 virus were successfully analyzed by the LAMP assay using tubes containing wax-sealed SGI and the prototype instrument, indicating that the method has the advantage of easy set-up (no extra components need to be added into the LAMP mixture for detection), high sensitivity (fluorescent intercalator), low background (detected at 75 degrees C) and no cross-contamination (closed-tube). Therefore, the novel LAMP detection, coupled with the instrument has the potential to be a diagnostic tool for a number of clinical applications in hospitals as well as on-site screening of pathogenic agents.