Convenient Polymerase Chain Reaction Research Articles

Simple innovative adaptor to improve genome walking with convenient PCR

BackgroundVarious polymerase chain reaction (PCR)-based methods have been applied for the development of genome walking (GW) technique. These methods which could be based on the application of restriction enzymes or primers have various efficiencies to identify the unknown nucleotide sequences. The present study was conducted to design a new innovative double-strand adaptor using MAP30 gene sequence of Momordica charantia plant as a model to improve genome walking with convenient PCR. ResultsThe adaptor was designed using multiple restriction sites of Hind III, BamH I, EcoR I, and Bgl II enzymes with no restriction site in a known sequence of the MAP30 gene. In addition, no modification was required to add phosphate, amine, or other groups to the adaptor, since restriction enzyme digestion of double-strand adaptor provided the 5′ phosphate group. Here, preparation of the phosphate group in the genomic DNA of the plant digestion with restriction enzymes was performed followed by ligation with digested adaptor containing 5′ phosphate group. ConclusionPCR was done to amplify the unknown sequence using MAP30 gene-specific primer and adaptor primer. Results confirmed the ability of the technique for successful identification of the sequence. Consequently, a newly designed adaptor in the developed technique reduced the time and cost of the method compared to the conventional genome walking; also, cloning and culturing of bacterial steps could be eliminated.

Species identification of trichomonads and associated coinfections in dogs with diarrhea and suspected trichomonosis

Trichomonads have been infrequently reported in the feces of dogs where their pathogenicity remains uncertain. It is currently unknown whether Tritrichomonas foetus or Pentatrichomonas hominis is identified more commonly in dogs with trichomonosis or how often these infections are accompanied by concurrent enteric infectious agents. The objective of this study was to determine the identity of trichomonads present in a series of 38 unsolicited canine diarrheic fecal samples submitted for T. foetus diagnostic polymerase chain reaction (PCR) testing between 2007 and 2010. We also examined each fecal sample for an association of trichomonosis with concurrent infection using a convenient real-time PCR panel for nine gastrointestinal pathogens. P. hominis, T. foetus, or both were identified by PCR in feces of 17, 1, and 1 dogs respectively. Feces from the remaining 19 dogs were PCR negative for T. foetus, P. hominis and using broader-spectrum Trichomonadida primers. The total number and specific identities of concurrent enteropathogens identified did not differ between fecal samples from dogs that were or were not identified by PCR as infected with trichomonads. These results suggest that P. hominis infection is more frequently identified than T. foetus infection in diarrheic dogs with trichomonosis and that concurrent enteropathogen infection is common in this population.

Multiplex polymerase chain reaction method to detect Cyclospora, Cystoisospora, and Microsporidia in stool samples

Cyclospora, Cystoisospora, and Microsporidia are eukaryotic enteropathogens that are difficult to detect in stool samples because they require special stains and microscopy. We developed a multiplex polymerase chain reaction (PCR) reaction with 4 primer sets to amplify Cyclospora cayetanensis, Cystoisospora belli, Enterocytozoon bieneusi, and Encephalitozoon intestinalis. Detection of the amplicon is through specific probes coupled to Luminex beads. Sensitivity of the assay was evaluated using Encephalitozoon intestinalis spores and revealed detection of 10 1 spores spiked into stool. No cross-reactivity was observed. We evaluated the assay on diarrheal specimens from Thailand, Tanzania, Indonesia, and the Netherlands that had been previously tested by microscopy, and the assay yielded 87–100% sensitivity and 88–100% specificity. Microscopy-negative/PCR-positive samples had lower Luminex values, suggesting they were true but with lower burden infections. In summary, this is a convenient single PCR reaction that can detect Cyclospora, Cystoisospora, and Microsporidia without the need for cumbersome microscopic analysis.

Comprehensive assessment of the TCRBV repertoire in small T-cell samples by means of an improved and convenient multiplex PCR method

Overall diversity of the T-cell receptor (TCR) repertoire can be regarded as a recapitulatory signature of a host's immunocompetence status. We aimed to establish a time- and cost-saving multiplex polymerase chain reaction (PCR) method for determining the TCR repertoire of conventional alphabeta T cells in small T-cell samples. The method estimates the length distribution of the complementarity-determining regions 3 (CDR3) of beta variable (BV) gene segments (TCRBV repertoire) by multiplex PCR, followed by fluorescent run-off reactions to visualize BV-BC and/or BV-BJ rearrangements. Run-off products are separated on a capillary sequencer and subsequently analyzed with GeneScan or Genotyper programs. Detection-limit studies with normal T cells, KMS27 cells, and regulatory T cells were carried out to evaluate sensitivity and reproducibility. Head-to-head comparison of the method with conventional immunoscope assay has shown that it is a time- and cost-saving approach to characterize TCRBV and TCRBJ repertoires, including the presence of oligoclonal T cells in samples containing as few as 1 x 10(5) T cells. We have developed a multiplex PCR method that allows comprehensive assessment of the TCRBV repertoire at the BV-BC and BV-BJ levels, and saves a considerable amount of time, reagents, and cell input.

An Emulsion System Based on a Chip Polymerase Chain Reaction

In this paper we describe a novel method for detecting many DNA fragments through efficient amplification by using an emulsion system based on “on-chip” PCR instead of conventional multiplex polymerase chain reaction (PCR). During the preparation of on-chip PCR, a set of primers were immobilized on a slide and other sets were in an emulsion system. Different emulsion phase primers and other related PCR components were dispersed in different droplets of the emulsion system, and then, due to the thermal instability of emulsion droplets, they would be released onto the surface of the slide after preheating in the first PCR step. To test the above method, we used plasma DNAs from pregnant women who was carrying a male fetus for gender identification. Four different Y chromosome DNA fragments were selected. Results showed that different DNA fragments could be simultaneously amplified with satisfactory results. It is suggested that a simple, convenient and inexpensive on-chip PCR method has been developed.

Polymerase Chain Reaction Method to Detect Canis Materials by Amplification of Species-Specific DNA Fragment

Rapid identification of mammal materials in feeding stuffs and food is essential for effective control of a potential source of pathogens, such as those that cause bovine spongiform encephalopathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a canis-specific mitochondrial DNA sequence in foodstuffs and food. The amplified canis-specific PCR product was a 213 base pair band from the D-loop DNA fragment of mitochondria, a high copy gene which should improve the possibility of amplifying template molecules of adequate size among the degraded DNA fragments brought about by heat denaturation. The specificity of this method was confirmed by 8 canis blood DNA samples (from different breeds of dog) and 9 noncanis animal blood DNA samples (bovine, sheep, porcine, chicken, fish, donkey, rabbit, deer, horse). This method was able to detect the presence of canis material in foodstuffs and in food mixtures even when the concentration of canis-derived meat was reduced to 0.05%. Furthermore, it did not appear to be affected by prolonged heat treatment. This method was developed for detection of canis materials in feeding stuffs, and occasionally for medical jurisprudence detection of canis-derived materials.

Multiplex Polymerase Chain Reaction Method for Detection of Bovine Materials in Foodstuffs

Rapid identification of bovine materials in animal foodstuffs is essential for effective control of a potential source of bovine spongiform encephalophathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a bovine-specific genomic DNA sequence in foodstuffs. Simultaneously the assay assessed the DNA quality of the experiment system by amplification of a highly conserved eucaryotic DNA region of the 18-S ribosomal gene, helping to check the reliability of the test result. The amplified bovine-specific PCR product was a genomic DNA fragment of lactoferrin, a low copy gene that was different from a commonly used bovine-specific mitochondria sequence for identification of bovine materials. The specificity of this method was confirmed by the absence of detectable homologous PCR product using reference foodstuff samples that lacked bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method could detect the presence of bovine material in foodstuffs when the samples contained > 0.02% bovine-derived meat and bone meal. Furthermore, it was not affected by prolonged heat treatment. The specificity, convenience, and sensitivity of this method suggest that it can be used for the routine detection of bovine-derived materials.

Mathematical considerations of competitive polymerase chain reaction

Reverse transcriptase polymerase chain reaction (PCR) is used frequently to monitor gene expression. It is generally regarded as a qualitative technique, although refinements have been made to improve quantification. The object of this study was to develop competitive PCRs to allow reliable quantification of the rat T cell cytokines interferon-γ (IFN-γ), interleukin-2 (IL-2) and interleukin-4 (IL-4). Truncated constructs of cDNA for these cytokines were prepared using appropriate pairs of standard and specially constructed primers designed to allow subsequent co-amplification of the purified competitor construct and the target cDNA. A high resolution capillary electrophoresis (CE) system was used for PCR product detection. The performance of the system was compared with a mathematical model that describes and predicts the exponential nature of the PCR reaction. Co-amplification of the competitor and target were achieved. A high level of resolution and accuracy was achieved using CE to detect and quantify the PCR products. The rates of generation of the respective products conformed closely but not exactly to the predictions of the mathematical model. The competitive PCRs estimated initial numbers of target cDNA within 1.1–5.0-fold relative to the amount of starting material as assessed by conventional spectrophotometric absorbance prior to dilution and amplification. A convenient and flexible competitive PCR strategy has been developed with accurate resolution of products and reliable quantification. Assay variability was far less than biological variability likely to be encountered in experiments investigating immunological responses in rats or other animals.

Detection of Chlamydia trachomatis in urine by polymerase chain reaction: Preliminary results

Detection of Chlamydia trachomatis in specimens of urine from men with acute urethritis by means of a rapid and convenient polymerase chain reaction (PCR)-based kit is reported. Results of the PCR were compared with those obtained by an enzyme immunoassay (EIA). Of 52 first-voided urine specimens, 22 (42.3%) were positive for C trachomatis, as determined by both PCR and EIA; 4 (7.7%) were positive by PCR and negative by EIA, and only 1 (1.9%) was negative by PCR and positive by EIA. The remaining 25 (48.1%) were negative by both PCR and EIA. These findings suggest that PCR assay is suitable for detection of C trachomatis in urine and may be more sensitive than EIA. Further studies are indicated.