Polymerase Chain Reaction Procedure Research Articles

Development of a Polymerase Chain Reaction Procedure for Detection of Chicken and Turkey Parvoviruses

Development of a Polymerase Chain Reaction Procedure for Detection of Chicken and Turkey Parvoviruses

Development of a Polymerase Chain Reaction Procedure for Detection of Chicken and Turkey Parvoviruses

Comparative sequence analysis of six independent chicken and turkey parvovirus nonstructural (NS) genes revealed specific genomic regions with 100% nucleotide sequence identity. A polymerase chain reaction (PCR) assay with primers targeting these conserved genome sequences proved to be highly specific and sensitive to detecting parvoviruses in experimentally infected chickens. In a nationwide survey, a total of 138 field enteric samples from poultry flocks were tested by PCR for parvovirus presence. Of the tested chicken samples that were collected in 54 farms, 77% showed the presence of parvovirus, while 78% of the turkey samples that were received from 29 farms were parvovirus positive. For the first time, our data clearly demonstrate that parvoviruses are widely distributed in commercial poultry flocks in the United States. The high prevalence of parvovirus infection in birds from enteric disease-affected flocks suggests a potential role of these viruses in the etiology of enteric disease of poultry. Phylogenetic analyses comparing NS gene segments showed that most of the chicken and turkey parvovirus isolates formed separate phylogenetic groups. These findings suggest that the chicken and turkey parvoviruses might have diverged from a common ancestor and have subsequently undergone host-specific adaptation.

Brain and lung cryptococcoma and concurrent corynebacterium pseudotuberculosis infection in a goat: a case report

A four-year-old male goat with a history of neurological disorder was euthanized. It presented uncommon nodules in the brain and lungs associated with multiple abscesses, predominantly in the spleen and liver. Histological examination of brain and lung sections revealed yeast forms confirmed to be Cryptococcus gattii after a combination of isolation and polymerase chain reaction (PCR) procedures. Moreover, Corynebacterium pseudotuberculosis infection was diagnosed by PCR of samples from the lung, spleen and liver. The present report highlights the rare concurrent infection of C. gatti and C. pseudotuberculosis in an adult goat from Sao Paulo state, Brazil, and indicates the necessity of surveillance in the treatment of goats with atypical pulmonary infections associated with neurological disorders.

Two‐step multiplex polymerase chain reaction improves the speed and accuracy of genotyping using DNA from noninvasive and museum samples

Many studies in molecular ecology rely upon the genotyping of large numbers of low-quantity DNA extracts derived from noninvasive or museum specimens. To overcome low amplification success rates and avoid genotyping errors such as allelic dropout and false alleles, multiple polymerase chain reaction (PCR) replicates for each sample are typically used. Recently, two-step multiplex procedures have been introduced which drastically increase the success rate and efficiency of genotyping. However, controversy still exists concerning the amount of replication needed for suitable control of error. Here we describe the use of a two-step multiplex PCR procedure that allows rapid genotyping using at least 19 different microsatellite loci. We applied this approach to quantified amounts of noninvasive DNAs from western chimpanzee, western gorilla, mountain gorilla and black and white colobus faecal samples, as well as to DNA from ~100-year-old gorilla teeth from museums. Analysis of over 45000 PCRs revealed average success rates of >90% using faecal DNAs and 74% using museum specimen DNAs. Average allelic dropout rates were substantially reduced compared to those obtained using conventional singleplex PCR protocols, and reliable genotyping using low (<25pg) amounts of template DNA was possible. However, four to five replicates of apparently homozygous results are needed to avoid allelic dropout when using the lowest concentration DNAs (<50pg/reaction), suggesting that use of protocols allowing routine acceptance of homozygous genotypes after as few as three replicates may lead to unanticipated errors when applied to low-concentration DNAs.

Bacterial Infection of Smad3/Rag2 Double-Null Mice with Transforming Growth Factor-β Dysregulation as a Model for Studying Inflammation-Associated Colon Cancer

Alterations in genes encoding transforming growth factor-beta-signaling components contribute to colon cancer in humans. Similarly, mice deficient in the transforming growth factor-beta signaling molecule, Smad3, develop colon cancer, but only after a bacterial trigger occurs, resulting in chronic inflammation. To determine whether Smad3-null lymphocytes contribute to increased cancer susceptibility, we crossed Smad3-null mice with mice deficient in both B and T lymphocytes (Rag2(-/-) mice). Helicobacter-infected Smad3/Rag2-double knockout (DKO) mice had more diffuse inflammation and increased incidence of adenocarcinoma compared with Helicobacter-infected Smad3(-/-) or Rag2(-/-) mice alone. Adoptive transfer of WT CD4(+)CD25(+) T-regulatory cells provided significant protection of Smad3/Rag2-DKO from bacterial-induced typhlocolitis, dysplasia, and tumor development, whereas Smad3(-/-) T-regulatory cells provided no protection. Immunohistochemistry, real-time reverse transcriptase-polymerase chain reaction, and Western blot analyses of colonic tissues from Smad3/Rag2-DKO mice 1 week after Helicobacter infection revealed an influx of macrophages, enhanced nuclear factor-kappaB activation, increased Bcl(XL)/Bcl-2 expression, increased c-Myc expression, accentuated epithelial cell proliferation, and up-regulated IFN-gamma, IL-1alpha, TNF-alpha, IL-1beta, and IL-6 transcription levels. These results suggest that the loss of Smad3 increases susceptibility to colon cancer by at least two mechanisms: deficient T-regulatory cell function, which leads to excessive inflammation after a bacterial trigger; and increased expression of proinflammatory cytokines, enhanced nuclear factor-kappaB activation, and increased expression of both pro-oncogenic and anti-apoptotic proteins that result in increased cell proliferation/survival of epithelial cells in colonic tissues.

A novel molecular pattern for methicillin-resistant Staphylococcus aureus in Milwaukee, WI clinical isolates

Methicillin-resistant Staphylococcus aureus is a significant problem in healthcare settings around the world. To effectively track different strains, we used a number of genetic procedures. In this study, 252 clinical isolates from the Milwaukee, WI, area were analyzed by the multiplex polymerase chain reaction (PCR) method to determine which of the described types were present. Most strains were categorized into one of the known groups, although types III and IIIA strains were not found. An unusual variant was found in 17% of the samples. This variant (v1) contains an 800-base pair amplimer that was created from a unique configuration of 2 primers from different sets in the multiplex PCR procedure. Although superficially similar to a type I pattern, sequencing of the amplimer demonstrated that it is identical to a sequence from the USA300 strain, a type IV strain. It is suggested that this variant may have resulted from acquisition of an IS431 element near the dcs region. There is potential for misidentification of strains because of slight genetic alterations such as this one. Statistical analysis using a χ 2 contingency table demonstrated that amoxicillin/clavulanic acid resistance was significantly lower in types I and IA when compared with the other types, whereas erythromycin resistance was significantly lower in type I when compared with other types. However, there was not a significant difference for any of the new variants.

Simulated childbirth injuries in an inbred rat strain

Vaginal distension (VD) in outbred rats has been shown to decrease urethral resistance, as well as increase the expression of the stem cell-homing chemokine, monocyte chemotactic factor 3 (MCP-3), but not stromal derived factor 1 (SDF-1). The aim of this study was to determine if similar responses are induced by VD in an inbred rat strain. Forty female Lewis rats underwent VD or sham VD followed by leak point pressure (LPP) testing 4 or 10 days later. Ten additional rats served as controls. The urethra and vagina were then dissected for histology. To examine chemokine expression, eight additional rats underwent VD with organs harvested immediately or 1 day after the procedure for reverse transcriptase polymerase chain reaction (RT-PCR) of MCP-3 and SDF-1. Four age-matched rats served as controls. Four days after VD, LPP was significantly lower in VD rats (14.3 +/- 1.6 cm H(2)O) than controls (18.7 +/- 1.3 cm H(2)O). Ten days after VD, LPP in both VD (19.7 +/- 2.6 cm H(2)O) and sham (18.4 +/- 1.3 cm H(2)O) groups was not significantly different from controls. Urethral histology demonstrated marked disruption and atrophy of smooth and striated muscle in VD rats compared to shams and controls. RT-PCR yielded a 25-fold significant increase in expression of urethral MCP-3 immediately following VD. SDF-1 was significantly decreased in the urethra and vagina immediately after VD and in the bladder 24 hr after VD. VD in Lewis rats produces functional, histological and molecular results similar to that of outbred rats. This model could be utilized in future studies investigating cellular transplant methods of improving urethral function.

An indirect ELISA and a PCR technique for the detection of Grouper ( Epinephelus marginatus ) mislabeling

An indirect enzyme-linked immunosorbent assay (ELISA) and a multiplex polymerase chain reaction (PCR) procedure was applied for the detection of Grouper (Epinephelus marginatus) mislabelling in the fish market. An indirect ELISA (microtiter-plate format) using two monoclonal antibodies (3D12 and 1A4) was assayed and multiplex PCR performed using species-specific primers of the 5S rDNA gene for the rapid authentication of grouper. A total of 70 commercial fish fillet samples, collected from local markets and supermarkets, labelled as grouper were analysed: 12 of the 70 samples were confirmed to be Grouper. The PCR technique permitted the detection of Nile Perch (Lates niloticus) in the commercial fillet samples, which was not possible using ELISA. The results suggest that both ELISA and PCR are specific and reliable tools for the detection of Grouper mislabelling/adulteration and the accurate implementation of traceability for successful regulatory food controls.

Development of 1480 nm Photothermal High-Speed Real-Time Polymerase Chain Reaction System for Rapid Nucleotide Recognition

The polymerase chain reaction (PCR) is a key technology used in genome-based biological analysis; however, requests have been made to shorten the operation time for emergency tests such as medical diagnostics, and countermeasures against bioterrorism. We have developed a novel rapid real-time PCR system using the direct absorption of an IR laser beam by water droplets as the heating device. The advantage of this system is that only the target water droplet was heated photothermally without transmitting any heat to the surroundings, which is important for the production of fast thermal cycle intervals. The system consists of a fluorescent microscope, an oil chamber with a set of water droplets lined up at the bottom, a 1480 nm IR laser unit, which is absorbed by water and can be focused on the droplets on the stage of the microscope, and an image intensifier to quantify the PCR reaction within a water droplet by measuring the change of fluorescent intensity. Using the system, we examined the PCR procedure under the following conditions: initial heating to 95 °C, maintaining this temperature for 10 s, and the suggested here and in similar places throughout 50 cycles of 1 s at 95 °C for denaturation and 3 s at 60 °C for annealing/extension. The temperature increase and decrease between the two temperatures 95 and 60 °C, were within 1 and 0.8 s respectively, i.e., 32 K/s, which is 1.5 times faster than the conventional heat conduction-based system. Rapid PCR amplification was observed successfully by the rise change in the sigmoidal curvature of fluorescent intensity, and the procedure was accomplished within 3.5 min, including the initial heating and complete 50 PCR cycles. The results indicate that the direct absorption-based heating of water droplets photothermally could give us a faster temperature chnage than the conventional heat-conduction-based systems such as Peltier heating/cooling.

Do Heat Shock Protein 70-2 and Interleukin-6 Genes Contribute to Exercise-Induced Increases in Creatine Kinase?

Strenuous exercise can provoke a marked increase in creatine kinase (CK), an indicator of muscle damage. Why some persons develop extremely high levels of CK in response to exercise is unknown, but clearly genetics serves a role. Two genes postulated to be important in the CK response to exercise include heat shock protein 70-2 (HSP70-2) and interleukin-6 (IL-6). PURPOSE: To assess the influence of selected single nucleotide polymorphisms (SNP) in the IL-6 (-C174G) and HSP70-2 (+A1538G) genes on baseline and exerciseinduced increases in CK. METHODS: Healthy, physically active participants (n=138) underwent an exercise step test for 5 min. with a backpack weighted at 30% of their body weight, followed by 15 squats. Blood was taken before, immediately after, 48 and 72 hrs after the step test for assessment of serum CK and isolation of DNA. SNPs in the HSP70-2 and IL-6 genes were evaluated by standard polymerase chain reaction procedures in blood samples. RESULTS: Participants were categorized into two groups based on their CK response. High responders (HR) had = 230 μU/L increases in CK at 48 or 72 hours post exercise relative to baseline; all others were normal responders (NR): 19 of 138 (14.5%) were HR. The percent of persons with the GG genotype for the IL-6 SNP was significantly higher in HR (68.4%) relative to NR (42.0%; p = 0.03). In addition, serum CK levels at 48 and 72 hours were significantly higher in GG as compared to C+ genotypes (IL-6), and GG as compared to AG genotypes (HSP70-2), but baseline CK levels did not differ when analyzed by ANOVA.TableCONCLUSIONS: These data suggest that HSP70-2 and IL-6 genes may serve a role in individual susceptibility to exertional muscle injury, as judged by CK responses to exercise. Supported by USU Grant R091CE.

Prevalence on beef carcasses of Mycobacterium avium subsp. paratuberculosis DNA

Fifty samples were collected from each of skinned and dressed carcasses, from each of culled beef breeding cows and fed beef cattle < 18 months old at two beef packing plants A and B, and from culled dairy cows at a packing plant C. The 450 samples were collected by swabbing an area of about 1000 cm 2 in the anal region of each carcass. DNA extracted from each swab was tested for the IS900 and F57 sequences of the Mycobacterium avium subsp. paratuberculosis (MAP) genome by two stage, nested polymerase chain reaction (PCR) procedures. An internal amplification control (IAC) was detected in 45 or more of each group of 50 DNA preparations. IS900 and F57 were detected in some IAC-positive preparations from all and all but one of the groups of carcasses, respectively. Of the IAC-positive preparations in each group, between 6 and 54% were positive for IS900, and between 4 and 20% were positive for F57. When preparations were tested by single stage, quantitative PCR procedures, IS900 was detected in two samples but F57 was detected in none. The MAP DNA on carcasses was probably derived from small numbers of MAP from the environment that contaminated the animals' hides.

130 Activated Innate Immune System in Irritable Bowel Syndrome?

The discovery of antimicrobial peptides has extended our knowledge of non-specific defence mechanisms. In addition to the physical barrier, the intestinal epithelium contributes to host defence by producing antimicrobial peptides to limit access to enteric bacteria and other microorganisms.1 The production of inducible antimicrobial peptides offers an initial rapid defence response of epithelial cells against invading microbes.2 Human β-defensin-2 (HBD-2) was the first inducible human antimicrobial protein discovered.3 It can be induced by probiotic microorganisms and pro-inflammatory cytokines.4 Recent results suggest that HBD-2 is expressed in active intestinal inflammation, especially in ulcerative colitis.5 Thus far, the expression of β-defensins has been quantified in the intestinal mucosa using the polymerase chain reaction procedure. Our aim was to evaluate faecal measurements of HBD-2 in patients with active …

Direct real-time PCR detection of Plum pox virus in field surveys in Ontario

A new procedure is described for the detection of Plum pox virus (PPV) in samples from large-scale field tests. The new direct real-time polymerase chain reaction (drtPCR) procedure was based on crude supernatants collected from peach (Prunus persica) leaves macerated in a buffer that was specially developed for this purpose and named “direct pathogen extract buffer.” Specific TaqMan primers and probes were designed for PPV detection: two sets specific to PPV D and M strains, respectively, and one for universal detection of PPV strains D, M, C, EA, and W. These primer and probe sets can be used singly or for duplex differentiation of D and M strains. Using Prunus spp. tissue infected with 30 known fruit tree viruses, the universal primer and probe set correctly identified PPV-infected and PPV-free samples, with no nonspecific cross-reactions. Based on endpoint analysis of the drtPCR reaction, a threshold cyle value of 36 was suggested as the maximum threshold to declare a sample positive for PPV. A comparative analysis comparing drtPCR and ELISA using 12 200 field samples revealed that drtPCR was approximately 100- to 1000-fold more sensitive than ELISA and was able to detect PPV at an earlier stage of infection than ELISA. The drtPCR is a valuable tool for PPV diagnosis and may also be applicable to other studies, including pathogen population dynamics and vector transmission efficiency.

A Simple and Rapid PCR-based Procedure for Identifying Homozygous Transgenic Rice Plants

We present a simple and rapid method for screening second-generation transgenic rice plants (T1) to identify homozygous plants. The plasmid (pfd11) used for rice transformation contains a partially deleted cytochrome c gene (cyc) for comparing with the endogenous cyc for copy number. After polymerase chain reaction (PCR) amplification of a segment of the cyc in transgenic rice DNA followed by agarose gel electrophoresis, two specific bands are obtained. The upper band represents the endogenous cyc, and the lower band represents the partially deleted cyc in the transgene. The first-generation plants (T0) that harbor a single copy of the transgene are selected based on the fact that the density of the lower band is half as dense as the upper band. Next, only plants harboring a single copy of the transgene are advanced to the second generation (T1). The same PCR procedure is used again, and homozygous T1 plants are easily identified from samples in which the intensity of the two bands is the same.

Defect-Aware High-Level Synthesis and Module Placement for Microfluidic Biochips

Recent advances in microfluidics technology have led to the emergence of miniaturized biochip devices, also referred to as lab-on-a-chip, for biochemical analysis. A promising category of microfluidic biochips relies on the principle of electrowetting-on-dielectric, whereby discrete droplets of nanoliter volumes can be manipulated using an array of electrodes. As chemists adapt more bioassays for concurrent execution on such ldquodigitalrdquo droplet-based microfluidic platforms, system integration, design complexity, and the need for defect tolerance are expected to increase rapidly. Automated design tools for defect-tolerant and multifunctional biochips are important for the emerging marketplace, especially for low-cost, portable, and disposable devices for clinical diagnostics. We present a unified synthesis method that combines defect-tolerant architectural synthesis with defect-aware physical design. The proposed approach allows architectural-level design choices and defect-tolerant physical design decisions to be made simultaneously. We use a large-scale protein assay and the polymerase chain reaction procedure as case studies to evaluate the proposed synthesis method. We also carry out simulations based on defect injection to evaluate the robustness of the synthesized biochip designs.

High‐throughput Procedure for Single Pollen Grain Collection and Polymerase Chain Reaction in Plants

Single pollen grain polymerase chain reaction (PCR) has succeeded in several species, however only limited numbers of pollen grains were involved due to difficulties in pollen isolation and lysis. This has limited its application in genetic analysis and mapping studies in plants. A high-throughput (HT) procedure for collecting and detecting genetic variation in a large number of individual pollen grains by PCR is reported. The HT procedure involved the collection of individual pollen grains by a pair of special forceps and the lysis of pollen grains in a heated alkali/detergent solution followed by neutralization with a tris-ethylenediamine tetraacetic acid (TE) buffer. These resulting template solutions yielded PCR reactions involving the 5S ribosomal RNA intergenic spacers, randomly amplified polymorphic DNA, and simple sequence repeats markers. Using this procedure, one person with experience could collect and process up to 288 single pollen grain PCR reactions per day. The method worked well on sugarcane, corn, Miscanthus spp., snap bean, sorghum, and tomato. The ability to collect and conduct PCR on individual pollen grains on a large scale offers a new approach to genetic analyses and mapping studies in an easily controllable environment with a considerable cost reduction. The method will also significantly benefit studies in species that are difficult subjects for classical genetic research.

Heteroplasmies Detected in an Amplified Mitochondrial DNA Control Region from a Small Amount of Template

When mitochondrial DNA (mtDNA) heteroplasmies are detected, they often confound forensic identification, especially if they are the result of poor biological sampling. In this study, we determined the ratio of heteroplasmy in samples that were amplified from a very small amount of template mtDNA or a few cells using a highly sensitive nested polymerase chain reaction (PCR) procedure and a direct sequencing analysis. As a result, more than half of the detected sequences (i.e., 17/20, 15/20, and 14/20) showed homoplasmy derived from a variation in the heteroplasmy proportion when only 10 copies of template mtDNA samples were amplified and analyzed. Additionally, with products amplified from one or several white blood cells (WBCs), several previously undetected heteroplasmies were detected. These results indicate the risks associated with using highly sensitive mtDNA techniques in forensic investigations because of the variable proportions of heteroplasmy or nucleotide substitutions that can possibly be detected from a very small biological sample.

Use of the lambda Red recombinase system to rapidly generate mutants in Pseudomonas aeruginosa

BackgroundThe Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in various bacteria and fungi. The procedure consists of electroporating a polymerase chain reaction (PCR) fragment that was obtained with a 1- or 3-step PCR protocol and that carries an antibiotic cassette flanked by a region homologous to the target locus into a strain that expresses the lambda Red recombination system.ResultsThis system has been modified for use in Pseudomonas aeruginosa. Chromosomal DNA deletions of single genes were generated using 3-step PCR products containing flanking regions 400–600 nucleotides (nt) in length that are homologous to the target sequence. A 1-step PCR product with a homologous extension flanking region of only 100 nt was in some cases sufficient to obtain the desired mutant. We further showed that the P. aeruginosa strain PA14 non-redundant transposon library can be used in conjunction with the lambda Red technique to rapidly generate large chromosomal deletions or transfer mutated genes into various PA14 isogenic mutants to create multi-locus knockout mutants.ConclusionThe lambda Red-based technique can be used efficiently to generate mutants in P. aeruginosa. The main advantage of this procedure is its rapidity as mutants can be easily obtained in less than a week if the 3-step PCR procedure is used, or in less than three days if the mutation needs to be transferred from one strain to another.

Improved normalization of real-time reverse transcriptase polymerase chain reaction data using an external RNA control

No golden standard exists for normalization of real-time reverse transcriptase polymerase chain reaction (RT PCR) data and procedures used are often not validated. Numerous studies have indicated that current approaches are inadequate. Here, we report the development of an external RNA control approach. It is the first to add external RNA to tissue on a per unit weight basis, and we demonstrate its accuracy, suitability, and necessity in experiments involving severe physiological challenges. We utilized the approach to examine the expression of the internal RNA control genes (reference genes) β-actin, cyclophilin A, and glyceraldehyde 3-phospate dehydrogenase in brain and heart of normoxic and anoxic crucian carp (Carassius carassius). The internal RNA control genes differed significantly in expression in experimental groups, especially in heart. We also demonstrate that the external RNA control approach provides a more accurate normalization of target genes. For example, it revealed a 2.5-fold increase in the expression of the stress-response gene HSC70, which was not detected using β-actin or geNorm. Further, we demonstrate and discuss the need for using the optimized and standardized external RNA control protocol reported. Collectively, our data suggest that the normalization of real-time RT PCR data is considerably improved by adding an external RNA control to the samples.

Development of a Real Time RT-PCR Assay for Detecting Genetically Different Pepino Mosaic Virus Isolates

Over a period of few years, Pepino mosaic virus (PepMV) has become one of the most impor- tant viral pathogen in tomato production worldwide. So far, five PepMV genotypes (EU, LP, CH2, US2 and US1) have been detected. A real time reverse transcription polymerase chain reaction (RT- PCR) procedure, using the fluorescence dye SYBR Green was developed for a rapid and reliable de- tection of genetically diverse Pepino mosaic virus isolates. This procedure was used for the detection and identification of PepMV in both Solanum lycopersicum and Nicotiana benthamiana species. The melting temperature (Tm) for members of a particular strain was very similar, with a host effect that did not hinder strain identification. Under optimal reaction conditions, sensitivity of the detection was as low as 100 fg of viral RNA from infected plants. This level of sensitivity indicated that the real time RT-PCR developed in the present study could be used for routine plant health assays.