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Journal of Applied Genetics Feb 2017PCR has become an essential tool in biological science. However, researchers often encounter problems with difficult targets, inhibitors accompanying the samples, or PCR... (Review)
Review
PCR has become an essential tool in biological science. However, researchers often encounter problems with difficult targets, inhibitors accompanying the samples, or PCR trouble related to DNA polymerase. Therefore, PCR optimization is necessary to obtain better results. One solution is using modified DNA polymerases with desirable properties for the experiments. In this article, PCR troubleshooting, depending on the DNA polymerase used, is shown. In addition, the reasons that might justify the need for modification of DNA polymerases, type of modifications, and links between modified DNA polymerases and PCR efficiency are described.
Topics: DNA-Binding Proteins; DNA-Directed DNA Polymerase; Mutation; Polymerase Chain Reaction; Recombinant Fusion Proteins
PubMed: 27796943
DOI: 10.1007/s13353-016-0371-4 -
Parasites & Vectors Nov 2022Leishmania infections span a range of clinical syndromes and impact humans from many geographic foci, but primarily the world's poorest regions. Transmitted by the bite... (Review)
Review
Leishmania infections span a range of clinical syndromes and impact humans from many geographic foci, but primarily the world's poorest regions. Transmitted by the bite of a female sand fly, Leishmania infections are increasing with human movement (due to international travel and war) as well as with shifts in vector habitat (due to climate change). Accurate diagnosis of the 20 or so species of Leishmania that infect humans can lead to the successful treatment of infections and, importantly, their prevention through modelling and intervention programs. A multitude of laboratory techniques for the detection of Leishmania have been developed over the past few decades, and although many have drawbacks, several of them show promise, particularly molecular methods like polymerase chain reaction. This review provides an overview of the methods available to diagnostic laboratories, from traditional techniques to the now-preferred molecular techniques, with an emphasis on polymerase chain reaction-based detection and typing methods.
Topics: Animals; Humans; Female; Leishmaniasis; Psychodidae; Leishmania; Polymerase Chain Reaction; Phlebotomus
PubMed: 36335408
DOI: 10.1186/s13071-022-05524-z -
Scientific Reports Jul 2023The procedure illustrated in this paper represents a new method for transcriptome analysis by PCR (Polymerase Chain Reaction), which circumvents the need for elimination...
The procedure illustrated in this paper represents a new method for transcriptome analysis by PCR (Polymerase Chain Reaction), which circumvents the need for elimination of potential DNA contamination. Compared to the existing methodologies, our method is more precise, simpler and more reproducible because it preserves the RNA's integrity, does not require materials and/or reagents that are used for elimination of DNA and it also reduces the number of samples that should be set up as negative controls. This novel procedure involves the use of a specifically modified primer during reverse transcription step, which contains mismatched bases, thus producing cDNA molecules that differ from genomic DNA. By using the same modified primer in PCR amplification, only cDNA template is amplified since genomic DNA template is partially heterologous to the primer. In this way, amplification by PCR is unaffected by any potential DNA contamination since it is specific only for the cDNA template. Furthermore, it accurately reflects the initial RNA concentration of the sample, which is prone to changes due to various physical or enzymatic treatments commonly used by the current methodologies for DNA elimination. The method is particularly suitable for quantification of highly repetitive DNA transcripts, such as satellite DNA.
Topics: DNA, Complementary; Reverse Transcription; Polymerase Chain Reaction; DNA; RNA; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 37454173
DOI: 10.1038/s41598-023-38383-4 -
Biosensors May 2022Polymerase chain reaction (PCR) is limited by the long reaction time for point-of-care. Currently, commercial benchtop rapid PCR requires 30−40 min, and this time is...
Polymerase chain reaction (PCR) is limited by the long reaction time for point-of-care. Currently, commercial benchtop rapid PCR requires 30−40 min, and this time is limited by the absence of rapid and stable heating and cooling platforms rather than the biochemical reaction kinetics. This study develops an ultrafast PCR (<3 min) platform using flow-through microchannel chips. An actin gene amplicon with a length of 151 base-pairs in the whole genome was used to verify the ultrafast PCR microfluidic chip. The results demonstrated that the channel of 56 μm height can provide fast heat conduction and the channel length should not be short. Under certain denaturation and annealing/extension times, a short channel design will cause the sample to drive slowly in the microchannel with insufficient pressure in the channel, causing the fluid to generate bubbles in the high-temperature zone and subsequently destabilizing the flow. The chips used in the experiment can complete 40 thermal cycles within 160 s through a design with the 56 µm channel height and with each thermal circle measuring 4 cm long. The calculation shows that the DNA extension speed is ~60 base-pairs/s, which is consistent with the theoretical speed of the Klen Taq extension used, and the detection limit can reach 67 copies. The heat transfer time of the reagent on this platform is very short. The simple chip design and fabrication are suitable for the development of commercial ultrafast PCR chips.
Topics: DNA; Microfluidics; Polymerase Chain Reaction
PubMed: 35624604
DOI: 10.3390/bios12050303 -
Clinical Infectious Diseases : An... Oct 2015Aspergillus polymerase chain reaction (PCR) was excluded from the European Organisation for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG)... (Comparative Study)
Comparative Study Review
BACKGROUND
Aspergillus polymerase chain reaction (PCR) was excluded from the European Organisation for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) definitions of invasive fungal disease because of limited standardization and validation. The definitions are being revised.
METHODS
A systematic literature review was performed to identify analytical and clinical information available on inclusion of galactomannan enzyme immunoassay (GM-EIA) (2002) and β-d-glucan (2008), providing a minimal threshold when considering PCR. Categorical parameters and statistical performance were compared.
RESULTS
When incorporated, GM-EIA and β-d-glucan sensitivities and specificities for diagnosing invasive aspergillosis were 81.6% and 91.6%, and 76.9% and 89.4%, respectively. Aspergillus PCR has similar sensitivity and specificity (76.8%-88.0% and 75.0%-94.5%, respectively) and comparable utility. Methodological recommendations and commercial PCR assays assist standardization. Although all tests have limitations, currently, PCR is the only test with independent quality control.
CONCLUSIONS
We propose that there is sufficient evidence that is at least equivalent to that used to include GM-EIA and β-d-glucan testing, and that PCR is now mature enough for inclusion in the EORTC/MSG definitions.
Topics: Antigens, Fungal; Aspergillosis; Aspergillus; Galactose; Humans; Immunoenzyme Techniques; Mannans; Polymerase Chain Reaction; Quality Control; Sensitivity and Specificity; beta-Glucans
PubMed: 26113653
DOI: 10.1093/cid/civ507 -
International Journal of Molecular... Jul 2023DNA polymerases have played an important role in molecular biology for several years and are frequently used for polymerase chain reaction (PCR); hence, there is an...
DNA polymerases have played an important role in molecular biology for several years and are frequently used for polymerase chain reaction (PCR); hence, there is an increasing interest in developing a convenient method for preparing DNA polymerase for routine use in laboratories. We developed a method using () that expresses thermostable DNA polymerase directly in the PCR without purification. The gene was transformed into and expressed. After overnight incubation and washing, -expressing DNA polymerase (EcoliTaq) was used as the DNA polymerase without purification. EcoliTaq showed activity comparable to that of commercial DNA polymerase and remained stable for 3 months. With a high-pH buffer containing 2% Tween 20 and 0.4 M trehalose, EcoliTaq facilitated direct PCR amplification from anticoagulated whole blood samples. EcoliTaq exhibited good performance in allele-specific PCR using both purified DNA and whole blood samples. Furthermore, it proved to be useful as a DNA polymerase in hot-start PCR by effectively minimizing non-specific amplification. We developed a simple and cost-effective direct and hot-start PCR method in which EcoliTaq was used directly as a PCR enzyme, thus eliminating the laborious and time-consuming steps of polymerase purification.
Topics: Taq Polymerase; Escherichia coli; Polymerase Chain Reaction; DNA; DNA Replication
PubMed: 37511160
DOI: 10.3390/ijms241411405 -
Archives of Razi Institute Aug 2023is the main cause of glanders as a dangerous contagious zoonosis disease that is mostly observed in single-hoofed animals, especially horses. Modern molecular...
is the main cause of glanders as a dangerous contagious zoonosis disease that is mostly observed in single-hoofed animals, especially horses. Modern molecular techniques have been recently employed to improve epidemiology for identifying and searching for strains of this bacterium at different times and locations. Due to the unknown number of circulating strains and lack of preventive methods, glanders is still observed in the form of epidemics. The present study aimed to evaluate six field isolates plus two laboratory strains of and using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. All the isolates and strains were microbially cultured in the glycerol nutrient and glycerol agar media. The individually grown colonies of the bacterium were used in the biochemical tests. The DNA of isolates was extracted by boiling, and the PCR-RFLP test was conducted on their genome. Finally, the bacterium was injected into guinea pigs to induce the Straus reaction. The biochemical assays (or bioassays) confirmed the isolates as . The PCR-RFLP assay demonstrated a product for with a length of 650 bp. Nevertheless, 250 and 400 bp were produced for . The swollen scrotum pointed to the occurrence of the Straus reaction. The PCR-RFLP is a proper differential diagnosis technique for ; moreover, it is a suitable method for differentiating between and . This technique can detect in a short time with high precision and sensitivity.
Topics: Horses; Animals; Guinea Pigs; Burkholderia mallei; Glanders; Polymorphism, Restriction Fragment Length; Glycerol; Burkholderia pseudomallei; Polymerase Chain Reaction; Horse Diseases
PubMed: 38226390
DOI: 10.32592/ARI.2023.78.4.1305 -
Nature Communications Aug 2023DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases....
DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases. Analysis of DNA methylation patterns has until now been dependent on either a chemical or an enzymatic pre-treatment, which are both time consuming procedures and potentially biased due to incomplete treatment. We present a qPCR technology, EpiDirect®, that allows for direct PCR quantification of DNA methylations using untreated DNA. EpiDirect® is based on the ability of Intercalating Nucleic Acids (INA®) to differentiate between methylated and unmethylated cytosines in a special primer design. With this technology, we develop an assay to analyze the methylation status of a region of the MGMT promoter used in treatment selection and prognosis of glioblastoma patients. We compare the assay to two bisulfite-relying, methyl-specific PCR assays in a study involving 42 brain tumor FFPE samples, revealing high sensitivity, specificity, and the clinical utility of the method.
Topics: Polymerase Chain Reaction; DNA; DNA Methylation; Temperature; Oligonucleotides; CpG Islands
PubMed: 37620381
DOI: 10.1038/s41467-023-40873-y -
BioTechniques Apr 2017New applications are driving the need for faster, higher fidelity PCR. Nathan Blow looks at some clever modifications taking PCR to the next level.
New applications are driving the need for faster, higher fidelity PCR. Nathan Blow looks at some clever modifications taking PCR to the next level.
Topics: Humans; Polymerase Chain Reaction
PubMed: 28403804
DOI: 10.2144/000114531 -
Malaria Journal Jul 2020Plasmodium knowlesi and Plasmodium vivax are the predominant Plasmodium species that cause malaria in Malaysia and play a role in asymptomatic malaria disease...
BACKGROUND
Plasmodium knowlesi and Plasmodium vivax are the predominant Plasmodium species that cause malaria in Malaysia and play a role in asymptomatic malaria disease transmission in Malaysia. The diagnostic tools available to diagnose malaria, such as microscopy and rapid diagnostic test (RDT), are less sensitive at detecting lower parasite density. Droplet digital polymerase chain reaction (ddPCR), which has been shown to have higher sensitivity at diagnosing malaria, allows direct quantification without the need for a standard curve. The aim of this study is to develop and use a duplex ddPCR assay for the detection of P. knowlesi and P. vivax, and compare this method to nested PCR and qPCR.
METHODS
The concordance rate, sensitivity and specificity of the duplex ddPCR assay were determined and compared to nested PCR and duplex qPCR.
RESULTS
The duplex ddPCR assay had higher analytical sensitivity (P. vivax = 10 copies/µL and P. knowlesi = 0.01 copies/µL) compared to qPCR (P. vivax = 100 copies/µL and P. knowlesi = 10 copies/µL). Moreover, the ddPCR assay had acceptable clinical sensitivity (P. vivax = 80% and P. knowlesi = 90%) and clinical specificity (P. vivax = 87.84% and P. knowlesi = 81.08%) when compared to nested PCR. Both ddPCR and qPCR detected more double infections in the samples.
CONCLUSIONS
Overall, the ddPCR assay demonstrated acceptable efficiency in detection of P. knowlesi and P. vivax, and was more sensitive than nested PCR in detecting mixed infections. However, the duplex ddPCR assay still needs optimization to improve the assay's clinical sensitivity and specificity.
Topics: Diagnostic Tests, Routine; Humans; Malaysia; Plasmodium knowlesi; Plasmodium vivax; Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 32650774
DOI: 10.1186/s12936-020-03314-5