Single-nucleotide polymorphisms (SNPs) and single-nucleotide mutations result from the substitution of only a single base. The SNP or mutation can be relevant to disease susceptibility, pathogenesis ...of disease, and efficacy of specific drugs. It is important to detect SNPs or mutations clinically. Methods to distinguish/detect SNPs or mutations should be highly specific and sensitive. In this regard, polymerase chain reaction (PCR) has provided the necessary analytical performance for many molecular analyses. PCR-based methods for SNP/mutation detection are broadly categorized into two types-(1) polymorphic or mutant allele-directed specific analysis using primers matched with substituted nucleotide or using oligonucleotides to block or clamp the nontargeted template, and (2) melting curve analysis, which is combined with the real-time PCR techniques using hydrolysis probes, hybridization probes, or double-stranded DNA-binding fluorescent dyes. Innovative and novel approaches as well as technical improvements have made SNP- or mutation-detection methods increasingly more sophisticated. These advances include DNA/RNA preparation and subsequent amplification steps, and miniaturization of PCR instruments such that testing may be performed with relative ease in clinical laboratories or as a point-of-care test in clinical settings.
•We tested three treatments to reduce quantitative PCR inhibition.•Ten-fold dilution and column purification treatments reduced inhibition the most.•Column purified samples underestimated DNA ...concentrations by 25%.•DNA concentration estimates were most precise for column purified samples.•Purified samples were most sensitive and accurate for relative concentrations.
Isolation of environmental DNA (eDNA) is an increasingly common method for detecting presence and assessing relative abundance of rare or elusive species in aquatic systems via the isolation of DNA from environmental samples and the amplification of species-specific sequences using quantitative PCR (qPCR). Co-extracted substances that inhibit qPCR can lead to inaccurate results and subsequent misinterpretation about a species’ status in the tested system. We tested three treatments (5-fold and 10-fold dilutions, and spin-column purification) for reducing qPCR inhibition from 21 partially and fully inhibited eDNA samples collected from coastal plain wetlands and mountain headwater streams in the southeastern USA. All treatments reduced the concentration of DNA in the samples. However, column purified samples retained the greatest sensitivity. For stream samples, all three treatments effectively reduced qPCR inhibition. However, for wetland samples, the 5-fold dilution was less effective than other treatments. Quantitative PCR results for column purified samples were more precise than the 5-fold and 10-fold dilutions by 2.2× and 3.7×, respectively. Column purified samples consistently underestimated qPCR-based DNA concentrations by approximately 25%, whereas the directional bias in qPCR-based DNA concentration estimates differed between stream and wetland samples for both dilution treatments. While the directional bias of qPCR-based DNA concentration estimates differed among treatments and locations, the magnitude of inaccuracy did not. Our results suggest that 10-fold dilution and column purification effectively reduce qPCR inhibition in mountain headwater stream and coastal plain wetland eDNA samples, and if applied to all samples in a study, column purification may provide the most accurate relative qPCR-based DNA concentrations estimates while retaining the greatest assay sensitivity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
43.
Digital PCR: A brief history Morley, Alexander A
Biomolecular detection and quantification,
09/2014, Volume:
1, Issue:
1
Journal Article
Peer reviewed
Open access
Digital PCR for quantification of a target of interest has been independently developed several times, being described in 1990 and 1991 using the term "limiting dilution PCR" and in 1999 using the ...term "digital PCR". It came into use in the decade following its first development but its use was cut short by the description of real-time PCR in 1996. However digital PCR has now had a renaissance due to the recent development of new instruments and chemistry which have made it a much simpler and more practical technique.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Objective To establish a method for qualitative detection of the presence or absence of all KIR genes by quantitative polymerase chain reaction(Q-PCR). Methods Based on the polymorphism of ...high-resolution level KIR alleles in Chinese population and the IPD-KIR database, KIR gene-specific primers were designed to amplify all the 16 KIR genes and 2DS4-Normal and 2DS4-Deleted subtypes by Q-PCR. Meanwhile, one negative control and one positive control specific amplifying human growth hormone (HGH) gene fragment were set to monitor the false positive and false negative results in PCR amplification, respectively. A total of 302 samples with known KIR genotype previously identified by KIR PCR-SSP commercial kit were randomly selected for blind inspection to verify the reliability of KIR Q-PCR method established by authors. Results The results of 300 samples detected by our KIR Q-PCR method were consistent with the known results, but two samples showed inconsistent results. One sample was negative for 2DS5 by Q-PCR b
Reverse Transcription PCR (RT-PCR) and Real Time Reverse Transcription PCR (rRT-PCR) are methods that can be used to detect VHSV (Viral Hemorrhagic Septicemia Virus) on freshwater and seawater fish. ...rRT-PCR is more widely used than the RT-PCR method because of several advantages such as requiring shorter time and fewer stages. Both methods have their own advantages and disadvantages, so it is necessary to compare the two methods in detecting the presence of VHSV in marine ornamental fish. The research was carried out with 3 steps: sensitivity test, specificity test, and sample test. The sensitivity test showed that the smallest dilution concentration that could be detected by both methods was the same at 10-3 with 100 copies in the 77bp band. Specificity tests on both methods showed that VHSV primers used are specific. Sample tests in both methods showed the same results in each of the samples tested. The results of sensitivity, specificity and sample tests conducted on RT-PCR and rRT-PCR methods showed the same results (positive) thus both methods can be used to detect the VHSV in marine ornamental fish.
Various attempts to amplify an AQP11 cDNA from tissues of the spiny dogfish (
) were made. Two pairs of deoxy-inosine-containing degenerate primers were designed based on conserved amino acid ...sequences from an AQP11 alignment. These primers yielded some faint bands from gill cDNA that were sequenced. Blast searches with the sequences showed they were not AQP11. An elasmobranch AQP11 nucleotide sequence alignment was produced to identify conserved regions to make further degenerate primers. One primer pair produced a short 148 bp fragment showing particularly strong amplification in gill and intestine. It was sequenced and represented a piece of the AQP11 gene. However, as the fragment may have resulted from contaminating genomic DNA (in total RNA used to make cDNA), 5' and 3' RACE were performed to amplify the two ends of the putative cDNA. Furthermore, 5' and 3' RACE amplifications depend on the presence of a 5' cap nucleotide and a poly A tail, respectively on the putative AQP11 mRNA. Hence, successful amplification was only possible from cDNA and not genomic DNA. Nested RACE amplifications were performed using gill and intestinal RACE cDNA, but none of the DNA fragments sequenced were AQP11. Consequently, the spiny dogfish AQP11 gene may represent a pseudogene.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The increased interest in global food fraud has led to the development of a number of advanced methods, among which real-time polymerase chain reaction (PCR) currently plays an integral role in food ...authentication. However, the lack of standard parameters for the development and validation of real-time PCR methods hampers their utilization across different laboratories and conditions, leading to inconsistent results.
This review summarizes and assesses different methods presented throughout a large number of scientific papers, including DNA extraction, primer design, and quantification (or qualification) as well as parameters for the development and validation of real-time PCR methods in food analysis.
Key Findings and Conclusions: Inhibitors in DNA extracts can cause decreased PCR sensitivity and false negative results; thus, the use of PCR inhibition and amplification controls (e.g., the 18S ribosomal RNA gene) is essential for obtaining accurate real-time PCR results. In quantitative real-time PCR methods, the results obtained using species-specific systems need to be normalized by using reference systems for the improvement of their accuracy. Therefore, this review will provide researchers with a beneficial guide for the development of real-time PCR methods in a harmonious manner and contribute to an enhanced applicability of the methods developed.
•Different methods and criteria used for real-time PCR for food analysis are reviewed.•Use of various PCR inhibition and amplification controls is discussed in detail.•Different quantification and normalization methods are compared and assessed in depth.•Method acceptance parameters are suggested for reliable real-time PCR results.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Turkey was the largest rainbow trout producer of the European countries in 2016, and the reason for this production is mainly attributed to its egg and fry production. Flavobacterium psychrophilum ...cause the highest rates of mortality in the starting to feeding stages of the fish. In the present study, twenty‐five F. psychrophilum isolates recovered from rainbow trout, coruh trout and brook trout were analysed by RAPD‐PCR, ERIC‐PCR, REP‐PCR and PCR‐RFLP, including 16S rRNA, gyrA and gyrB gene regions and PCR‐based serotyping method. The PCR‐based molecular analysis showed that the isolates could not be differentiated exactly according to isolation source and geographical region. Most isolates were of type‐1 and type‐2, and some of them were of type‐0 and type‐3; in addition, one isolate showed a unique serotype. The combined analysis results showed that F. psychrophilum isolates discriminated as five different genotypes and all isolates were successfully discriminated based on host.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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