Polymerase Chain Reaction Conditions Research Articles

Harnessing multiplex polymerase chain reaction assay for convenient and simultaneous differentiation of testudinis carapax et plastrum from trionycis carapax

Background: Testudinis Carapax et Plastrum (TCP, Guijia) and Trionycis Carapax (TC, Biejia), which made from the shell of Chinemys reevesii (CR) and Pelodiscus sinensis (PS), were two kinds of widely used animal-derived Traditional Chinese medicine (TCM). Some cheap substitutes such as the carapace and plastron of Trachemys scripta (TS), Mauremys sinensis (MS), or Apalone ferox (AF) shells obtained from restaurants are sometimes used, which expose the public health to a high risk and cause unfair competitions in the market. Objective: The objective of the study was to develop a multiplex polymerase chain reaction (PCR) approach to simultaneously differentiate five Chelonia species and identify adulteration of two natural products. Materials and Methods: Five novel species-specific primers were designed for CR, TS, MS, PS, and AF, followed by optimization of PCR conditions and validation for specificity and sensitivity. Then, a deliberate mixture was analyzed to verify the capability of adulteration detection. Finally, it was used to examine commercial products. Results: The developed method proved to be highly specific and detection limit was 1 ng for all the species tested. Particularly, it was still applicable and reliable when processed products or deliberate adulteration were examined. Two batches of commercial raw TCP products were identified to be counterfeited by TS using the newly proposed approach. Conclusion: The newly proposed multiplex PCR method showed sufficient merits to be readily employed as a regular means to authenticate edible and medicinal TCM products made of TCP and TC.

Streptavidin Homologues for Applications on Solid Surfaces at High Temperatures.

One of the most commonly used bonds between two biomolecules is the bond between biotin and streptavidin (SA) or streptavidin homologues (SAHs). A high dissociation constant and the consequent high-temperature stability even allows for its use in nucleic acid detection under polymerase chain reaction (PCR) conditions. There are a number of SAHs available, and for assay design, it is of great interest to determine as to which SAH will perform the best under assay conditions. Although there are numerous single studies on the characterization of SAHs in solution or selected solid phases, there is no systematic study comparing different SAHs for biomolecule-binding, hybridization, and PCR assays on solid phases. We compared streptavidin, core streptavidin, traptavidin, core traptavidin, neutravidin, and monomeric streptavidin on the surface of microbeads (10-15 μm in diameter) and designed multiplex microbead-based experiments and analyzed simultaneously the binding of biotinylated oligonucleotides and the hybridization of oligonucleotides to complementary capture probes. We also bound comparably large DNA origamis to capture probes on the microbead surface. We used a real-time fluorescence microscopy imaging platform, with which it is possible to subject samples to a programmable time and temperature profile and to record binding processes on the microbead surface depending on the time and temperature. With the exception of core traptavidin and monomeric streptavidin, all other SA/SAHs were suitable for our investigations. We found hybridization efficiencies close to 100% for streptavidin, core streptavidin, traptavidin, and neutravidin. These could all be considered equally suitable for hybridization, PCR applications, and melting point analysis. The SA/SAH-biotin bond was temperature-sensitive when the oligonucleotide was mono-biotinylated, with traptavidin being the most stable followed by streptavidin and neutravidin. Mono-biotinylated oligonucleotides can be used in experiments with temperatures up to 70 °C. When oligonucleotides were bis-biotinylated, all SA/SAH-biotin bonds had similar temperature stability under PCR conditions, even if they comprised a streptavidin variant with slower biotin dissociation and increased mechanostability.

Validation of the Investigator 24plex QS Kit: a 6-dye multiplex PCR assay for forensic application in the Chinese Han population

The Investigator 24plex QS Kit (QIAGEN, Hilden, Germany) is a 6-dye fluorescent chemistry short tandem repeat (STR) polymerase chain reaction (PCR) amplification system that simultaneously amplifies 20 of the expanded Combined DNA Index System (CODIS) core STR loci, SE33, DYS391, and the standard sex-determining locus, amelogenin, as well as two special internal performance quality sensor controls (QS1 and QS2), which are included in the primer mix to check the PCR performance. This study was designed to be a pilot evaluation of this STR-PCR kit in a Chinese Han population regarding the PCR conditions, sensitivity, precision, accuracy, repeatability, reproducibility, and concordance; tolerance to PCR inhibitors; applicability to real “forensic-type” samples; species specificity; mixture, balance and stutter analyses, and utility in a population investigation. The exhaustive validation studies demonstrated that the Investigator 24plex QS system is accurate, sensitive and robust for STR genotyping. In addition, these genetic markers in the population data in our study indicated that they can also be useful for forensic identification and paternity testing in the Chinese Han population.

Evaluating novel effective primers to amplify heterozygous alleles of second, third and fourth exons of HLA-A; -B; -C; -DRB1 and -DQB1 loci using sequencing-based typing

Human leukocyte antigen (HLA) typing is one of the most crucial steps that determines the success of an organ transplant. However, HLA typing is a challenging task due to the diversity of HLA alleles, which is caused by high polymorphism of the region and high number of guanine and cytosine bases that limits the degree of amplification. Low resolution serology typing currently employed in Sri Lanka may fail to identify subtle differences in certain alleles, which may affect the long-term survival of the organ recipient. Therefore, a low cost, high-resolution DNA-based typing method for the HLA loci of Sri Lankans was developed based on polymerase chain reaction (PCR) amplification followed by Sanger sequencing, which is considered to be the gold standard for HLA typing. With minimised PCR bias and equal chances of amplifying all the alleles curated so far, a novel set of primers were designed to amplify the second and third exons of alleles in group specific PCR. To increase the resolution of alleles further, the fourth exon was also amplified using novel primers designed in this study and primers reported in the literature. Touchdown PCR and hot-start PCR were used to optimise PCR conditions so that non-specific amplifications are minimal. SBTengine® (version 3.12.0.2724) software was used in assigning the sequence chromatogram to the allele sequence. Seventeen new primers were designed in this study to ensure the amplification and identification of both alleles in heterozygous individuals that were previously unable to be identified using primers reported in the literature.

Synthetic gene as target to assess the sensitivity of PCR to detect Trichinella spp. larvae in meat from a non-endemic region.

Trichinellosis is a zoonotic disease exotic in Brazil but commonly found worldwide including South American countries like Argentina. International trading of swine meat needs an official Trichinella-free diagnosis commonly carried out by pepsin-HCl digestion of diaphragm tissue fragments followed by microscopic examination for the presence or absence of Trichinella larvae. The easiness of this diagnostic method allows it to be performed at slaughtering plants but, in contrast, it lacks sensitivity and does not allow species differentiation, which is fundamental for determining geographical and species distribution of different genotypes. In our study, we aimed to evaluate a highly sensitive diagnostic method based on the polymerase chain reaction (PCR) that would allow us to detect and classify different species of Trichinella. Thus, we designed a synthetic gene and selected five sets of primers targeting specific regions of the Trichinella genome. The synthetic gene was cloned into a plasmid and then used to optimize PCR conditions. Using our PCR, we were able to detect 0.001pg of the synthetic gene, which corresponded to 0.01 larvae. Then, we collected 175 samples of Suidae (domestic and wild boars) diaphragm fragments that were pooled into groups, digested with pepsin-HCl, and had the DNA extracted for analysis by PCR. The clinical samples evaluated were negative by PCR. Our results indicate that the PCR-based method might be a useful diagnostic method complementary to the pepsin-HCl digestion method currently in use, mostly in non-endemic areas.

X-Ray Repair Cross Complementing 4 (XRCC4) Gene Expressions as Biomarkers for Detection of Ionizing Radiation Exposure

Summary The present study aims at using the gene expression as biomarkers in the identification of the biological effects of low and high doses of ionizing radiation (X-ray) in white mice Mus musculus Balb/C, ages 4-6 weeks, weight 30-40 grams. Seventy- two white mice (36 males and 36 females) were divided into two groups; their whole body was exposed to 5 cGy (rad) and 100 cGy (rad) of X-ray radiation at a dose rate of 200 cGy/min, in addition to the control group. Total RNA was successfully isolated using Trizol method from blood and liver samples of mice after 6, 48 hours and 10 days of exposure to radiation as well as of the control group. The RNA concentration was determined spectrophotometrically by measuring their absorbance using nucleic acid and protein analyzer that dependent on the ratio A260/A280 of the wavelength which lead to the determination of RNA purity, it ranged from 1.79-2.1 in all mice groups. RNA integrity and quality were confirmed by agarose gel electrophoresis.Three bands such as 28s, 18s and 5s appeared in a visible manner. This study involved the reverse transcription (RT) of the RNA for the manufacture of complementary DNA (cDNA) using the polymerase chain reaction (PCR) for investigation on above -mentioned groups of animals. Complementary DNA was used in amplification of genes used in the present study, one type of specialized primers were selected for the gene as X-Ray repair cross complementing group 4 (XRCC4), which have a relation with ionizing radiation in addition to the primers for internal control (β-actin) gene. The optimal conditions for PCR were determined using a dye (SYBR ® Green 1).This should be done before using the device quantitative real time-PCR (QRT-PCR) in experiments. The products of replicated specialized primers for the genes concerned and the cDNA for the studied samples were electrophoretically separated in agarose gels .The banding profiles were visualized by ethidium bromide staining, as the molecular weight was 183 bp nitrogen-base pair for XRCC4 gene.The changes in the gene expression for the genes concerned were determined by measuring the quantitative levels of expression in the blood and liver samples of the group of mice after 6, 48 hours and 10 days of exposure to X-ray, in addition to the control group using the device QRT-PCR.The presence of significant reduction (p <0.05) in the amount of gene expression for the XRCC4 gene in samples of blood and liver from mice exposed to both doses of 5 cGy and 100 cGy after 6, 48 hours and 10 days of exposure to radiation. This gene was down-regulation after 6 hours in the blood samples of mice exposed to these doses compared to the control group. In contrast, there were to these finding, significant increases (p <0.05) in the amounts of gene expression of this gene in the liver tissues of mice exposed to both doses after 6 and 48 hours of exposure to radiation. This gene also showed up-regulation after 48 hours in the liver tissue of mice exposed to 5 cGy doses, while the up-regulation was appeared after 6 hours of exposure to 100 cGy dose of radiation.In conclusion, the results indicated that there is a possibility of using the changes in the level of XRCC4 gene expression as useful biomarkers for the detection of organism's exposure to ionizing radiation. .

Mating analyses of Trichophyton benhamiae offspring reveals linkage of genetic markers used in taxonomy.

Mating experiments were conducted with four clinical Trichophyton benhamiae isolates, genetically similar to the Trichophyton benhamiae CBS 112371, featuring the plus mating type and with two minus type strains. One minus type strain belonged to the white subgroup, and the other minus type strain, DSM 6916, showed genetic kinship to the yellow subgroup. Only two plus type strains were able to form mature, pigmented gymnothecia with DSM 6916. These two plus type strains demonstrated dark pigmentation and powdery mycelium on Takashio agar, whereas the other three strains exhibited a low degree of pigmentation on the same medium. All five plus strains were able to mate with the minus type strain of their own white subgroup. Cultures from single ascospore isolates showed highly variable morphology and pigmentation. Three genetic markers (ITS, mating type, EF1 alpha) were analyzed in polymerase chain reaction (PCR) experiments with optimized primers and PCR conditions to discriminate between subgroups. Furthermore, RAPD-PCR was used to generate a DSM 6916-specific DNA-fragment which served as an additional genetic marker. Assessing the isolates with recombinant genotypes, it was found that three genetic markers behave like linked genes. The recombination of plus mating type went together with ITS, EF1 alpha and RAPD marker of the DSM 6916 parental strain and was most frequently isolated, whereas plus types recombinants in this case were completely missing. This shows a high imbalance in mating type distribution of recombinants.

Development of a species-specific polymerase chain reaction-based technology for authentication of asini corii colla and taurus corii colla

Background: Asini Corii Colla (ACC; donkey-hide glue) and Taurus Corii Colla (TCC; bovine-hide glue) are popular healthcare food supplements as well as well-recognized medicine in Traditional Chinese medicine clinics. Due to large demand of them and limited resources of their animal origins, the skin of other animals are often used to make their fake or adulterated products. Objective: In this study, we aimed to develop a species-specific polymerase chain reaction (PCR) approach to detect deoxyribonucleic acid (DNA) fragments of ACC and TCC for rapid authentication purpose. Materials and Methods: Four sets of novel species-specific primer were designed, and PCR conditions were optimized for the PCR assay, which was further validated for specificity and sensitivity. Then, deliberate mixture was analyzed to further verify the capability of adulteration detection by the developed PCR method. Finally, it was used to assess the authenticity of commercial products. Results: Four primers' sets were specific for amplification of extracted mitochondrial DNA of donkey, bovine, swine, and horse when the annealing temperature was 59°C, 59°C, 63°C, and 65°C, and 40 thermal cycles was performed. Under this optimized PCR conditions, 1 ng/μL of horse DNA template has been detected in the sensitivity test, with 0.1 ng/μL for donkey, bovine, and swine. The assay was capable of detecting spiked species at 10% level in premixed ACC or TCC samples. Conclusion: The newly developed PCR-based technology was specific and sensitive, and it was a convenient approach for the authentication of commercial ACC and TCC products.

SONiCS: PCR stutter noise correction in genome-scale microsatellites

Massively parallel capture of short tandem repeats (STRs, or microsatellites) provides a strategy for population genomic and demographic analyses at high resolution with or without a reference genome. However, the high Polymerase Chain Reaction (PCR) cycle numbers needed for target capture experiments create genotyping noise through polymerase slippage known as PCR stutter. We developed SONiCS-Stutter mONte Carlo Simulation-a solution for stutter correction based on dense forward simulations of PCR and capture experimental conditions. To test SONiCS, we genotyped a 2499-marker STR panel in 22 humpback dolphins (Sousa sahulensis) using target capture, and generated capillary-based genotypes to validate five of these markers. In these 110 comparisons, SONiCS showed a 99.1% accuracy rate and a 98.2% genotyping success rate, miscalling a single allele in a marker with low sequence coverage and rejecting another as un-callable. Source code and documentation for SONiCS is freely available at https://github.com/kzkedzierska/sonics. Raw read data used in experimental validation of SONiCS have been deposited in the Sequence Read Archive under accession number SRP135756. Supplementary data are available at Bioinformatics online.

F47. Screening of GLE1 mutations in Chinese amyotrophic lateral sclerosis patients

Introduction Amyotrophic lateral sclerosis (ALS) is a fatal disease involving both upper and lower motor neuron, resulting in a 3–5 year of survival from symptom onset caused by rapidly progressive paralysis and respiratory failure. It is etiologically heterogeneous. Approximately 90% of ALS cases are sporadic (SALS) with unknown causes. But The remaining 10% of patients have a family history (FALS) and harbor a causative gene mutation, reflecting genetic factor play an important role in the pathogenesis of ALS. To date, mutations in over 20 genes have been reported in ALS populations. Mutations in GLE1 are the causation of two fetal motoneuron diseases: lethal congenital contracture syndrome 1 (LCCS1) and lethal arthrogryposis with anterior horn cell disease (LAAHD). Recently, Kaneb et al. identifiedGLE1variants within sporadic and familial Caucasian ALS cases, supporting defects in RNA metabolism were associated with pathogenesis of ALS. However, GLE1 mutations have not been investigated in Chinese ALS patients so far. Accordingly, we evaluated whether GLE1 mutations harbored in ALS patients from mainland China. Methods The current study recruited probands of 20 ALS families (mean age of onset was 50.1 ± 10.9 years; 10 females, 10 males), 230 SALS patients (mean age of onset was 52.1 ± 11.9 years; 96 females, 134 males). All 16 exons (plus 50 bp of flanking introns) of GLE1 (NM_001003722.1) including the entire coding region were amplified using polymerase chain reaction (PCR). The reaction conditions of PCR were as follows: 5 min of denaturation at 95 °C, 8 cycles were used with 20 s at 94 °C, 20 s at 65 °C (decreasing by 1 °C with each cycle) and 30 s at 72 °C. 20 s at 94 °C, 20 s at 58 °C and 30 s at 72 °C for the subsequent 34 cycles, a final extension step at 72 °C for 5 min. PCR products were subjected to Sanger sequencing using ABI 3730 automated DNA-sequencing system (Applied Biosystems). The sequences were aligned with reference sequence using CodonCode Aligner. Results GLE1 mutation screening of 250 ALS cases revealed 4 synonymous genetic variants(c.444G > A, p. R148R; c.946C > A, p. R316R; c.1641T > C, p. Y547Y; c.2082C > T, P. S694S), which presented in dbSNP database. Bioinformatic analysis using ESE finder and Human Splicing Finder 3.0 showed that all the silent mutations did not affect transcription process. We did not identify any novel or reported rare variants in all 16 coding exons of GLE1from 250 Chinese ALS patients. Conclusion In summary, our results suggested that GLE1 mutations are not common in FALS and SALS of Chinese origin. Further study in larger and additional populations would be required to evaluate the role of GLE1 in ALS causation.

Primer Design Using Polymerase Chain Reaction for SNPs Analysis in SLC22A1 rs622342 Encoding OCT1 as Metformin Main Transporter

Organic Cation Transporter 1 (OCT1) is the primary transporter of metformin mainly located in hepatocytes, which plays an important role in metformin action to inhibit gluconeogenesis so as to reduce blood glucose. Genetic polymorphism of SLC22A1 that encodes OCT1, one of which is rs622342, has been widely reported and proven to decrease the antidiabetic effect of metformin. This study aimed to design primers and to obtain an optimum condition for polymerase chain reaction (PCR) process that can detect the genetic polymorphism of SLC22A1 rs622342. Primers were computationally designed in primer 3 webpage and analyzed with Primer BLAST and Oligo Analyzer. Optimization of PCR condition was conducted for temperatures of denaturation, annealing, and elongation as well as for the number of cycles in PCR process. Sensitivity test was performed on PCR condition using a variety of volumes and DNA template qualities undergoing multiple freeze-thaw cycles. The obtained pair, forward primer (5’- CAG AGA GAA TCA GTG AGC TGT G-3’) and reverse primer (5’- CCC AGG CTG GTC TTT TTA AG-3’), was proven to be capable of amplifying DNA sequence containing SNPs in rs622342 at 96°C denaturation, 60°C annealing, and 72°C elongation temperatures with a 30-cycle iteration. Such PCR condition could amplify DNA with 0.2 µL of template volume and 7 freeze-thaw cycles. Therefore, in addition to the selected primer pairs and PCR condition to analyze SNPs in rs622342, this study also recommends that the volume of DNA template having undergone multiple freeze-thawing be increased if the amplicon PCR products are unqualified.

Portable low-power thermal cycler with dual thin-film Pt heaters for a polymeric PCR chip.

Polymerase chain reaction (PCR) has been widely used for major definite diagnostic tool, but very limited its place used only indoor such as hospital or diagnosis lab. For the rapid on-site detection of pathogen in an outdoor environment, a low-power cordless polymerase chain reaction (PCR) thermal cycler is crucial module. At this point of view, we proposed a low-power PCR thermal cycler that could be operated in an outdoor anywhere. The disposable PCR chip was made of a polymeric (PI/PET) film to reduce the thermal mass. A dual arrangement of the Pt heaters, which were positioned on the top and bottom of the PCR chip, improved the temperature uniformity. The temperature sensor, which was made of the same material as the heater, utilized the temperature dependence of the Pt resistor to ensure simple fabrication of the temperature sensor. Cooling the PCR chip using dual blower fans enabled thermal cycling to operate with a lower power than that of a Peltier element with a high power consumption. The PCR components were electrically connected to a control module that could be operated with a Li-ion battery (12V), and the PCR conditions (temperature, time, cycle, etc.) were inputted on a touch screen. For 30 PCR cycles, the accumulated power consumption of heating and cooling was 7.3Wh, which is easily available from a compact battery. Escherichia coli genomic DNA (510bp) was amplified using the proposed PCR thermal cycler and the disposable PCR chip. A similar DNA amplification capability was confirmed using the proposed portable and low-power thermal cycler compared with a conventional thermal cycler.

Діагностика грибів роду Fusarium у сіянцях сосни звичайної методом полімеразної ланцюгової реакції

Найбільш поширеною та небезпечною хворобою сіянців хвойних порід є інфекційне вилягання, збудниками якого є гриби з родів Fusarium, Alternaria, Botrytis та інші. Актуальним завданням є розроблення чутливих і селективних методів ідентифікації патогену. Представлено методику детекції ДНК грибів роду Fusarium у тканинах сіянців сосни звичайної шляхом полімеразно-ланцюгової реакції із видоспецифічними праймерами. Розроблена методика дає змогу визначати 0,1 пг ДНК Fusarium sporotrichella на високому фоні ДНК сосни, що перевищує кількість ДНК патогену в десять тисяч разів. Методику апробовано на різновікових сіянцях сосни звичайної, вирощених у нестерильних умовах закритого ґрунту. Проведено діагностику тканин сіянців сосни, що вилягли, та візуально здорових рослин на присутність ДНК фузаріумів.

Zika virus RNA polymerase chain reaction on the utility channel of a commercial nucleic acid testing system.

Several countries have implemented safety strategies to reduce the risk of Zika virus (ZIKV) transmission through blood transfusion. These strategies have included nucleic acid amplification testing (NAT) of blood donations. In this study, a new real-time polymerase chain reaction (PCR) assay including internal control for the detection of ZIKV on the cobas omni Utility Channel (UC) on the cobas 6800 system is presented. PCR conditions and primer/probe concentrations were optimized on the LightCycler 480 instrument. Optimized conditions were transferred to the cobas omni UC on the cobas 6800 system. Subsequently, the limit of detection (LOD) in plasma and urine, genotype inclusivity, specificity, cross-reactivity, and clinical sensitivity were determined. The 95% LOD of the ZIKV PCR assay on the cobas 6800 system was 23.0 IU/mL (95% confidence interval [CI], 16.5-37.5) in plasma and 24.5 IU/mL (95% CI, 13.4-92.9) in urine. The assay detected African and Asian lineages of ZIKV. The specificity was 100%. The clinical concordance between the newly developed ZIKV PCR assay and the investigational Roche cobas Zika NAT test was 83% (24/29). We developed a sensitive ZIKV PCR assay on the cobas omni UC on the cobas 6800 system. The assay can be used for large-scale screening of blood donations for ZIKV or for testing of blood donors returning from areas with ZIKV to avoid temporal deferral. This study also demonstrates that the cobas omni UC on the cobas 6800 system can be used for in-house-developed PCR assays.

Surface Enhanced Raman Spectroscopy (SERS) for the Multiplex Detection of Braf, Kras, and Pik3ca Mutations in Plasma of Colorectal Cancer Patients.

In this paper, we discuss the use of a procedure based on polymerase chain reaction (PCR) and surface enhanced Raman spectroscopy (SERS) (PCR-SERS) to detect DNA mutations.Methods: This method was implemented by first amplifying DNA-containing target mutations, then by annealing probes, and finally by applying SERS detection. The obtained SERS spectra were from a mixture of fluorescence tags labeled to complementary sequences on the mutant DNA. Then, the SERS spectra of multiple tags were decomposed to component tag spectra by multiple linear regression (MLR).Results: The detection limit was 10-11 M with a coefficient of determination (R2) of 0.88. To demonstrate the applicability of this process on real samples, the PCR-SERS method was applied on blood plasma taken from 49 colorectal cancer patients to detect six mutations located at the BRAF, KRAS, and PIK3CA genes. The mutation rates obtained by the PCR-SERS method were in concordance with previous research. Fisher's exact test showed that only two detected mutations at BRAF (V600E) and PIK3CA (E542K) were significantly positively correlated with right-sided colon cancer. No other clinical feature such as gender, age, cancer stage, or differentiation was correlated with mutation (V600E at BRAF, G12C, G12D, G12V, G13D at KRAS, and E542K at PIK3CA). Visually, a dendrogram drawn through hierarchical clustering analysis (HCA) supported the results of Fisher's exact test. The clusters drawn by all six mutations did not conform to the distributions of cancer stages, differentiation or cancer positions. However, the cluster drawn by the two mutations of V600E and E542K showed that all samples with those mutations belonged to the right-sided colon cancer group.Conclusion: The suggested PCR-SERS method is multiplexed, flexible in probe design, easy to incorporate into existing PCR conditions, and was sensitive enough to detect mutations in blood plasma.

Prevalence and molecular identification of &lt;i&gt;Campylobacter&lt;/i&gt; species isolates from poultry and humans in Sokoto, Nigeria

Prevalence and molecular identification of Campylobacter species isolates from poultry and humans were conducted using culture, biochemical reaction and Polymerase Chain Reaction (PCR) techniques. A total of 798 (506 poultry and 292 human) samples were identified biochemically, out of which 312(39.1%) were positive for Campylobacter species. Campylobacter jejuni, C. coli and C. lari had 38 (23.8%) out of 160, 63(39.4%) out of 160, 59(36.9%) out of 160 prevalence rates, respectively in humans while 29(19.1%) out of 152, 79(52.0%) out of 152 and 44(28.9%) out of 152 were the rates for the species in the same order in poultry. Campylobacter isolates were kept at -20oC in 15% glycerol and 85% tryptone broth until used while some were identified 24hrs post isolation. Single and multiplex PCR were used to confirm the genus Campylobacter and three Campylobacter species, respectively. All the 130(100 stored and 30 fresh) isolates were members of the genus Campylobacter. The single PCR band view of stored isolates also revealed other bands in addition to 439 bp which is specific for the genus Campylobacter, while the fresh isolates had distinct bands at 439bp only. Multiplex PCR revealed 2(6.7%) out of 30 were positive for stored isolates out of 30, 1(50%) each for C. jejuni and C. Coli. However, 1 of the stored isolate was positive for both spp. On the other hand, 6(20.0%) of out 60 fresh isolates were positive, with 5(83.3%) and 1(16.7%) for C. jejuni and C. coli, respectively. The possibilities of improper identification using conventional method have been revealed in the study. PCR can identify Campylobacter species more accurately than biochemical method, though storage of isolates, integrity of extracted DNA and PCR conditions can affect result. However, the use of both methods should be encouraged in regular and effective surveillance of Campylobacter species in poultry and humans.Keywords: Campylobacter species, Humans, Molecular identification , Nigeria, Prevalence, Poultry, Sokoto

A multiplex PCR strategy to screen for known mutations in families with sudden cardiac death burden.

Ventricular tachyarrythmia occurring in ischemic heart disease, dilated/hypertrophic cardiomyopathies or rare monogenic mutations of cardiac ion channels or associated proteins belong to the most frequent causes of sudden cardiac death (SCD). In further decades, next generation sequencing and bioinformatic analysis will become the gold standard of SCD risk stratification. At the moment, Sanger-sequencing is still obligatory in genetic diagnosis. A multiplex polymerase chain reaction (PCR) assay detecting eight SCD mutations in one reaction-tube was developed. To test the general validity of the assay, it was used with 12 patients, who had one or two of the eight mutations (LMNA, p.V256V; SCN5A, p.R1583C; RYR2, p.G1885E; MYH7, V606M; DSG2, p.T335A; KCNJ8, p.S422L; MYBPC, p.E441K; TNNT2, A38V). Thereafter, we tested the multiplex assay in a real diagnostic environment within a high risk family of several past SCD cases. This method allows efficient discrimination of multiple mutations by allele-specific PCR with standard PCR conditions. It relies on obtaining a PCR product specific to the mutation or wildtype—using primers that have the 3′end base complementary to the DNA template site, i.e. a specific primer only permits amplification to take place when its 3′terminal nucleotide matches with its target sequence. The PCR products are further analyzed by length, with Tape Station®(Agilent Technologies, Germany), a high-fidelity capillary chromatography test. The novel multiplex PCR assay strategy could be a good additional test used for SCD risk stratification. Advantages of the test are high velocity and ease of implementation, low price and flexibility of application within cardiomyopathy families for screening purposes.

Glass-polytetrafluoroethylene-glass based sandwich microdevice for continuous-flow polymerase chain reaction and its application for fast identification of foodborne pathogens

A uniquely fabricated microdevice with a glass-polytetrafluoroethylene (PTFE)-glass (GPG) sandwich configuration was developed for the fast identification of foodborne pathogens by using continuous-flow polymerase chain reaction (PCR) followed by on-site fluorescence detection of PCR products. A PTFE tube was simply folded into a serpentine configuration and sandwiched between two glass slides to maintain a 2D planar structure. By using a PTFE tube, the microchannel has the advantage of seamlessness without the need for photolithography methods, and is less expensive and less time-consuming to fabricate than alternative devices. The non-porous and gas-impermeable PTFE tube ensured stable sample flow inside the microchannel. PCR conditions were optimized and evaluated by examining the effects of number of cycles and initial concentration of bacteria introduced. On-site detection of PCR products was performed using nucleic acid dye SYBR Green I. Using the GPG microdevice, Escherichia coli O157:H7 and Salmonella spp., two well-known foodborne pathogens, were amplified within 20min. Besides fast reaction, the simply fabricated microdevice demonstrated its potential as a portable device applicable for public healthcare diagnostics.

Multiplex quantification of Escherichia coli, Salmonella typhi and Vibrio cholera with three DNA targets in single reaction assay

Escherichia coli (E. coli), Salmonella typhi and Vibrio cholera harmful pathogens, which causes various diseases in humans. Rapid diagnosis of bacterial infection is an important for patient management and appropriate therapy during the early phase of the bacterial infected diseases. Among the existing techniques for identifying pathogens were less sensitive and time-consuming processes. In the present study total, 48 clinical 31 blood and 17 urine samples of patients suspected with the infections were collected from SVRR Hospital and used to detect the pathogens. Multiplex polymerase chain reaction (PCR) assay was set to design for the identification of Escherichia coli, Salmonella typhi and Vibrio cholera from the different clinical samples. Rapid diagnosis of Escherichia coli (E. coli), Salmonella and Vibrio cholera pathogens can be done with simultaneously in a single multiplex PCR assay by using specific primers with adjusted PCR conditions. Through this approach, the results represented with out of 31 blood samples 1–15 shows the positive with E. coli and remaining 14 only 11 were correlated with multiplex results of Vibrio cholera, remaining the urine samples all are positive with 17 samples correlate with the Salmonella typhi. Through the high specificity benefits of excellent sensitivity, with high resolution and reproducibility. This method of results proved and illustrates the best potential system for diagnosing the infectious disease with modern trendy.

Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method

We identified Alizarin Red S and other well known fluorescent dyes useful for the online detection of pyrophosphate in enzymatic assays, including the loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. An iterative screening was used for a selected set of compounds to first secure enzyme compatibility, evaluate inorganic pyrophosphate sensitivity in the presence of manganese as quencher and optimize conditions for an online detection. Of the selected dyes, the inexpensive alizarin red S was found to selectively detect pyrophosphate under LAMP and PCR conditions and is superior with respect to its defined red-shifted spectrum, long shelf life and low toxicity. In addition, the newly identified properties may also be useful in other enzymatic assays which do not generate nucleic acids but are based on inorganic pyrophosphate. Finally, we propose that our screening method may provide a blueprint for rapid screening of compounds for detecting inorganic pyrophosphate.