Specific Polymerase Chain Reaction Products Research Articles

Optimization of polymerase chain reaction for the identification of Roe deer, Saiga, and Siberian stag living in Kazakhstan.

Background and Aim:One of the reasons for the decline in the number of wild species of artiodactyls is poaching and the illegal trading of animal products. Molecular genetic identification of animals from a biological sample effectively proves poaching cases and illegal trade of animal products. This study aimed to develop a polymerase chain reaction (PCR) test that allows for species identification of artiodactyl animals that are most often subject to poaching.Materials and Methods:Genomic DNA was extracted from meat and blood samples of animals killed by poachers using commercial kits. Three pairs of primers were designed and used to amplify the cytochrome b gene fragment of Roe deer, Saiga antelope, and Siberian stag.Results:The proposed protocol allows amplification of specific PCR products of 542 bp with Roe deer DNA, 587 bp with Saiga DNA, and 525 bp with Siberian stag DNA. Specificity analysis showed no cross activity with DNA from other animal species. The detection limit of PCR ranged from 15.6 pg to 1.9 pg of DNA in 25 mL of the reaction mixture.Conclusion:Sequencing the amplified products and subsequent comparison with the corresponding reference sequence showed a similarity ranging from 99.99% to 100%. The PCR based on the developed primers demonstrated high sensitivity and specificity when using DNA from homogeneous and heterogeneous animals.

Seroprevalence and molecular characterization of Toxoplasma gondii infecting ruminants in the North-West of Egypt

Toxoplasma gondii is a coccidian parasite known for its heavy toll on people and livestock. It can cause abortion and a variety of congenital diseases. The current study aimed to examine some seroprevalence and molecular attributes of T. gondii obtained from ruminants in the North-West of Egypt. Specimens were random selected from five different locations in Alexandria and Matrouh governorates. A total of 483 blood samples, collected from 96 mixed flocks, were screened for anti-T. gondii IgG antibodies using enzyme-linked immunosorbent assay (ELISA). The seropositive results were then confirmed using polymerase chain reaction (PCR) primers for the B1 and P30 genes. Specific PCR products were selected for sequencing and alignment against the GenBank, where phylogeny has been examined using the maximum likelihood, neighbor-joining, and maximum parsimony in MEGA6. ELISA confirmed the presence of T. gondii in 188 of the investigated samples (38.92%), indicating a higher prevalence in camels (64.51%) and sheep (43.75%) as compared to goats (27.93 %) and cattle (13.46%). PCR confirmed the presence of T. gondii-specific sequences in 159 seropositive specimens, with homology between 98.3 and 100%. The genetic distances between the investigated variants ranged from 0.1 to 0.9, and 7 single nucleotide polymorphisms (SNPs), were identified in the examined T. gondii specimens. The camel T. gondii parasite, isolated from Matrouh, showed a 100% homology with the most dangerous reference strains of T. gondii-RH in the GenBank. Our results showed that B1 and P30-specific PCR could detect T. gondii in blood samples more accurately than ELISA. In addition, the statistical analysis of our data indicated that species, age, sex, and animal location were all risk factors for toxoplasmosis. These findings are likely to boost disease control and help contain the spread of T. gondii infections.

Accomplishment of one-step specific PCR and evaluated SELEX process by a dual-microfluidic amplified system.

One of the main obstacles for systematic evolution of ligands by exponential enrichment (SELEX) failure is the generation of a non-specific product, as selection-inherent amplification procedures tend to form by-products, which prevents the enrichment of target-binding aptamers. Herein, we reported a dual-microfluidic amplified system (dual-MAS) based on the real-time polymerase chain reaction (PCR) detection chip and the large volume PCR chip for one-step specific PCR and for evaluating the SELEX process. First, it is a simple method to accomplish analytical PCR and amplification PCR in one step, and the optimal number of cycles for generating the specific PCR product is the cycles when the slope of the linear amplification period of the real-time PCR curve begins to decrease. Second, the time used by the dual-MAS for generating a specific PCR product is reduced to 30 min, and the multi-functional dual-MAS can simultaneously evaluate the SELEX process by providing important information on the amounts of enriched sequences and the library diversity in every round of SELEX. In addition, pollution contamination and fragment loss can be significantly avoided in the closed chip. Last, the specific PCR product, the amounts of enriched sequences, and the library diversity can be obtained for every single SELEX in just 30 min. Compared with current methods, this system can reduce the time for generating a specific PCR product and SELEX, and it is easier to choose the optimal number of cycles for a specific PCR product. In a word, it is a sensitive, simple, and rapid strategy to improve the specificity of the PCR product and make the process of SELEX in a controlled way.

Cleaved amplified polymorphic sequences (CAPS) marker for identification of two mutant alleles of the rapeseed BnaA.FAD2 gene

Two mutants of winter rapeseed (Brassica napus L. var. oleifera) with an increased amount of oleic acid in seeds were created by chemical mutagenesis (HOR3-M10453 and HOR4-M10464). The overall performance of the mutated plants was much lower than that of wild-type cultivars. Multiple rounds of crossing with high-yielding double-low (“00”) cultivars and breeding lines having valuable agronomic traits, followed by selection of high oleic acid genotypes is then needed to obtain new “00” varieties of rapeseed having high oleic acid content in seeds. To perform such selection, the specific codominant cleaved amplified polymorphic sequences (CAPS) marker was used. This marker was designed to detect the presence of two relevant point mutations in the desaturase gene BnaA.FAD2, and it was previously described and patented. The specific polymerase chain reaction product (732 bp) was digested using FspBI restriction enzyme that recognizes the 5′-C↓TAG-3′ sequence which is common to both mutated alleles, thereby yielding band patterns specific for those alleles. The method proposed in the patent was redesigned, adjusted to specific laboratory conditions, and thoroughly tested. Different DNA extraction protocols were tested to optimize the procedure. Two variants of the CAPS method (with and without purification of amplified product) were considered to choose the best option. In addition, the ability of the studied marker to detect heterozygosity in the BnaA.FAD2 locus was also tested. Finally, we also presented some examples for the use of the new CAPS marker in the marker-assisted selection (MAS) during our breeding programs. The standard CTAB method of DNA extraction and the simplified, two-step (amplification/digestion) procedure for the CAPS marker are recommended. The marker was found to be useful for the detection of two mutated alleles of the studied BnaA.FAD2 desaturase gene and can potentially assure the breeders of the purity of their HOLL lines. However, it was also shown that it could not detect any other alleles or genes that were revealed to play a role in the regulation of oleic acid level.

Polymerase chain reaction-based ultrasensitive detection of HBV DNA via G-quadruplex selective iridium(III) complex luminescent probe

Polymerase chain reaction (PCR) is the gold standard for low-abundant DNA detection. Here, to expand the application of PCR with novel detecting methods, we developed a label-free fluorescent sensor for ultrasensitive and one-step detection of hepatitis B virus (HBV) DNA using the G-quadruplex selective iridium(III) complex luminescent probe. By using HBV DNA as the template with two hairpin structure primers that contained oxyethylene glycol tethers, PCR amplification occurred and generated numbers of specific PCR products with free G-quadruplex sequences at both ends. Such free G-quadruplex sequences can change into G-quadruplex structure with the help of K+, resulting in a strong luminescence intensity upon their binding with the G-quadruplex selective iridium(III) complex. The luminescence intensity increase was proportional to the concentration of PCR products, and indirectly related with HBV DNA concentration. Moreover, the utilization of the iridium(III) complex effectively improved the specificity of the sensor, while PCR paved the way for the ultrasensitive detection of DNA in the linear range of 3.0 fM to 800 pM, with a detection limit of 1.6 fM. Notably, this assay was successfully used to detect HBV DNA in normal and patient serum samples, indicating a potential application for biomolecular analysis.

First Report of Cotton Leafroll Dwarf Virus Infecting Cotton (Gossypium hirsutum) in Kansas

In the United States, cotton leafroll dwarf disease, caused by cotton leafroll dwarf virus (CLRDV, Polerovirus, Luteoviridae), was first reported from cotton (Gossypium hirsutum L.) fields in Alabama (Avelar et al. 2019) and later Mississippi (Aboughanem-Sabanadzovic et al. 2019) and Georgia (Tabassum et al. 2019). During a survey of cotton fields in Barber County, Kansas, in the 2019 growing season, cotton plants showed virus-like symptoms including leaf yellowing, discoloration, reddening, and short internodes with reduced or small boll sets. Disease incidence based on virus-like symptoms was ∼1 to 15% depending on the affected fields. Eleven symptomatic and seven apparently asymptomatic leaf samples from 18 independent cotton plants (cv. PhytoGen 210) were collected in commercial cotton fields and tested for the presence of the virus in the Plant Virology Lab at the University of Tulsa, Oklahoma. Total RNA was extracted from all 18 samples by the Spectrum Plant Total RNA Kit (Sigma-Aldrich) and was synthesized into complementary DNA (cDNA) using the Superscript IV Reverse Transcription Kit (ThermoFisher Scientific) according to the manufacturer’s instructions. The polymerase chain reaction (PCR) was performed with primers CLRDV3675F and Pol3982R as described previously (Sharman et al. 2015) to amplify a specific PCR product of 310 bp of the ORF3-5 fragment of the CLRDV. cDNA of CLRDV for the specific fragment was obtained from the Plant Disease Diagnostic Lab at Auburn University and was used as a positive control. A PCR product of the predicted size (310 bp) was obtained from seven symptomatic cotton samples and the positive control, whereas no bands were present in four symptomatic and seven asymptomatic samples, as well as the PCR negative control. RT-PCR was repeated twice with these samples and obtained the same results as above. The PCR products of all positive samples were directly sequenced, and sequence comparison showed 97 to 99% identity with the CLRDV-Alabama isolate (MN071395). To further confirm the presence of CLRDV in these samples, two additional pair of primers—AL674F and AL1407R for partial P0/P1 (Avelar et al. 2019) and RdRpF2 and RdRpR1 for partial RdRp (Aboughanem-Sabanadzovic et al. 2019)—were used to amplify the 694- and 770-bp product of the CLRDV genome, respectively. The expected PCR bands were obtained from all seven symptomatic cotton plants but none from asymptomatic samples. PCR products of 694 and 770 bp from two samples (KS-7 and KS-10 isolates) were purified, cloned into pGEM-T Easy vector (Promega), and transformed into Escherichia coli DH5α cells (New England Bio Labs). At least three clones were sequenced using the Applied Biosystem 3130 Genetic Analyzer at the University of Tulsa. Sequences obtained in this study for partial P0/P1and RdRP genes of KS-7 and KS-10 isolates were deposited in GenBank (MN809925 to 28). Nucleotide sequence comparisons and BLASTn analysis of partial P1/P0 (MN809927 to 28) showed 98 to 99% identity with corresponding sequences of Alabama isolates (MH883237 and MN071395) and 92 to 93% identity with typical and 91 to 92% identity with atypical CLRDV isolates from Argentina and Brazil. Similarly, partial RdRP genes (MN809925 to 26) showed 94 to 98% identity with the corresponding sequences of available CLRDV isolates from Mississippi (MK512759, MK512762 to 64) and Alabama (MN071395) and 93 to 96% identity with typical and 92 to 95% identity with atypical CLRDV isolates from Argentina and Brazil. This is the first report of CLRDV infection and occurrence in cotton fields in Kansas. Cotton acreage in Kansas has increased since 2018 by 40% (NASS 2018), and the presence of CLRDV could be a detrimental disease for cotton production. Therefore, further studies are needed, including other cotton-growing counties in Kansas, to determine the distribution, epidemiology, vector transmission, and population structure of this virus, which would be helpful in the management of CLRDV.

The Weedy Parasite Phelipanche aegyptiaca Attacks Brassica rapa var. rapa for the First Time in Israel

The Weedy Parasite <i>Phelipanche aegyptiaca</i> Attacks <i>Brassica rapa</i> var. <i>rapa</i> for the First Time in Israel

Double-Stranded DNA Fragments Bearing Unrepairable Lesions and Their Internalization into Mouse Krebs-2 Carcinoma Cells

Murine Krebs-2 tumor-initiating stem cells are known to natively internalize extracellular double-stranded DNA fragments. Being internalized, these fragments interfere in the repair of chemically induced interstrand cross-links. In the current investigation, 756 bp polymerase chain reaction (PCR) product containing bulky photoreactive dC adduct was used as extracellular DNA. This adduct was shown to inhibit the cellular system of nucleotide excision repair while being resistant to excision by this DNA repair system. The basic parameters for this DNA probe internalization by the murine Krebs-2 tumor cells were characterized. Being incubated under regular conditions (60 min, 24°C, 500 μL of the incubation medium, in the dark), 0.35% ± 0.18% of the Krebs-2 ascites cells were shown to natively internalize modified DNA. The saturating amount of the modified DNA was detected to be 0.37 μg per 106 cells. For the similar unmodified DNA fragments, this ratio is 0.73 μg per 106 cells. Krebs-2 tumor cells were shown to be saturated internalizing either (190 ± 40) × 103 molecules of modified DNA or (1,000 ± 100) × 103 molecules of native DNA. On internalization, the fragments of DNA undergo partial and nonuniform hydrolysis of 3' ends followed by circularization. The degree of hydrolysis, assessed by sequencing of several clones with the insertion of specific PCR product, was 30-60 nucleotides.

Comparison of Two Multiplex PCR Systems for Meat Species Authentication

Background: Meat species adulteration has become a problem of concern. This study aimed to compare two previously published multiplex Polymerase Chain Reaction (PCR) methods for meat species authentication. &#x0D; Methods: The primers used in the first multiplex PCR involved species-specific reverse primer for sheep, goat, cattle, pig, and donkey with universal forward primer. In the second multiplex PCR, the primers included species-specific forward and reverse primer for pork, lamb, ostrich, horse, and cow. The extracted DNA was then amplified with species-specific primers and with mix primers separately in the respective multiplex PCR.&#x0D; Results: The first multiplex PCR was accompanied with cross reactivity, whereas the second multiplex PCR was specific as expected for pork, lamb, ostrich, horse, and cow. The first set of multiplex PCR showed not always amplification of all species-specific DNAs with a mixture of DNA from mentioned animals. Regarding the second set of primers, the extracted DNA of different meat species was amplified with corresponding species primers as simplex PCR resulting in specific amplicons for species DNA prepared from sheep, ostrich, horse, pig, and cattle with the specific PCR products of 119, 155, 253, 100, and 311 bp, respectively.&#x0D; Conclusion: Based on the present investigation, we recommend the multiplex PCR with the second set of primers included species-specific forward and reverse primers for species authentication of five meat types, including pork, lamb, ostrich, horse, as well as cow.

Establishment of a species-specific primer pair for detecting Veillonella infantium based on the 70 kDa heat shock protein gene dnaK

Recently, Veillonella infantium was isolated from tongue biofilm of a Thai child and established as a novel Veillonella species. In this study, a species-specific primer was designed to identify V. infantium on the basis of the sequence of the 70 kDa heat shock protein (dnaK) gene of Veillonella infantium JCM 31738T (= TSD-88T). The primer pair generated a specific PCR (Polymerase Chain Reaction) product specific for V. infantium, but not for other oral Veillonella species. This specific primer pair could detect dnaK even from 1 pg of genomic DNA extracted from the V. infantium type strain. To validate the primer pair, a number of strains of Veillonella species were isolated from tongue biofilm of 3 Japanese children, DNA was isolated from each strain, and PCR was performed using species-specific primers. All oral Veillonella species except V. infantium were identified by one-step PCR method reported previously. Four kinds of Veillonella species were detected in these subjects. V. rogosae was detected in all subjects and the most predominant species with an average prevalence of 82%. However, V. infantium was detected in 2 of 3 subjects and it was the second most predominant species of oral Veillonella detected in these subjects with an average prevalence of 9.4%. V. infantium appears to coexist with other oral Veillonella species in tongue biofilm. This species-specific primer pair established in this study could be useful to detect V. infantium and support the study of Veillonella for oral health in the future.

Assessment of some genetic attributes in wheat (Triticum aestivum L.) using gene-specific molecular markers

Twenty-four wheat genotypes were characterized for the presence of three stress related genetic attributes using gene-specific molecular markers. Polymerase chain reaction (PCR) products of approximately 110 bp of 1RS rye chromosome fragment from 16 genotypes were obtained using specific primer pairs indicating the presence of translocation in these lines. The same genotypes were screened for the presence of dwarfing genes where 19 genotypes showed the presence of either the Rht-B1b or Rht-D1b allele having a semi-dwarf phenotype. Two of the genotypes (Kheri and Sufi) had wild type alleles in both the loci and two other genotypes showed the presence of double dwarf alleles (Rht-B1b + Rht-D1b). A 16.9 kDa HSP gene was characterized by validating a single-nucleotide polymorphism (SNP) linked with thermo tolerance in wheat. Thirteen of the 24 genotypes, which failed to amplify the specific PCR product due to the presence of an SNP, are expected to show tolerance for heat stress. Among the genotypes, Sonora-64, Balaka, Barkat, Aghrani and BARI Gom-24 (Prodip) tested positive for all the three markers evaluated (rye translocation, dwarfing genes and heat tolerance). These genotypes could be used to improve various stress tolerance attributes in wheat for future breeding programs.

A Rapid and Cost-Efficient Technique for Simultaneous/Duplex Detection of Listeria Monocytogenes and Escherichia Coli O157:H7 Using Real Time PCR

The increasing industrial interest in easy, economical and reliable methods has led to the development and application of DNA-based methods for the detection of microbial pathogens in food. In the present article, we describe the development of a cost-efficient EvaGreen-based real-time Polymerase chain reaction (PCR) technique for simultaneous and practical detection of Listeria monocytogenes (L. monocytogenes) and Escherichia coli O157:H7 (E. coli O157:H7) in the food matrix. EvaGreen-based simplex and duplex real-time PCR showed that specific PCR products were identified by melting curve analysis, and had a reproducible Tm of 77.00 ± 0.4C for L. monocytogenes and 85.60 ± 0.2 for E. coli O157:H7.The absolute detection limit of Eva Green-based simplex and duplex real-time PCR was 0.0001 ng/μL for DNA of both pathogens. The relative detection limit of the simplex and duplex technique was less than 10 cells/mL for the two pathogens. Practical Applications This study illustrates a rapid, effective and cheap identification method to simultaneously monitor two foodborne pathogens in food samples. The specificity and sensitivity of this method can be used to clearly identify these two foodborne pathogens in practical food samples based on the specific genes of the species. Hence, this detection method is applicable to surveillance measures for these two foodborne pathogens in the food production chain. Moreover, this technique is used by food control laboratories in a secure way.

Analysis of the inhibition of nucleic acid dyes on polymerase chain reaction by capillary electrophoresis

An integrated polymerase chain reaction (PCR) and capillary electrophoresis (CE) system can realize accurate quantification of the target PCR product by adding labeling dyes to the PCR reagents, because CE can discriminate all the subsequent nucleic acids, including the primers, non-specific and specific PCR products.

Development of novel chloroplast microsatellite markers for Ginkgo biloba.

Ginkgo biloba is considered to be a living fossil that can be used to understand the ancient evolutionary history of gymnosperms, but little attention has been given to the study of its population genetics, molecular phylogeography, and genetic resources assessment. Chloroplast simple sequence repeat (cpSSR) markers are powerful tools for genetic studies of plants. In this study, a total of 30 perfect cpSSRs of Ginkgo were identified and characterized, including di-, tri, tetra-, penta-, and hexanucleotide repeats. Fifteen of 21 designed primer pairs were successfully amplified to yield specific polymerase chain reaction products from 16 Ginkgo cultivars. Polymorphic cpSSRs were further applied to determine the genetic variation of 116 individuals in 5 populations of G. biloba. The results showed that 24 and 76% genetic variation existed within and among populations of this species, respectively. These polymorphic and monomorphic cpSSR markers can be used to trace the origin and evolutionary history of Ginkgo.

Circulating microRNAs: Understanding the Limits for Quantitative Measurement by Real‐Time PCR

Ever since the discovery of small, non-coding RNAs in circulation, microRNAs have gained significant attention as biomarkers or, in some instances, regulators of disease and/or associated complications. Until now, most articles published use real-time quantitative PCR (qPCR) for assessment of

Quantitative Real-Time PCR for Determination of Transgene in Callus of Jatropha curcas

Jatropha curcas is an important plant belonging to the family Euphorbiaceae which is a potential candidate for biofuel production. Genetic transformation protocol for J. curcas callus mediated by Agrobacterium tumefaciens were optimized using a pCAMBIA1303 plasmid which carries green fluorescent protein (GFP) gene as a reporter. Results obtained were based on the highest percentage of GFP expression which was observed three days post-transformation. Immersion of callus into 1×105 cfu ml-1 (OD600nm 0.6) of A. tumefaciens LBA4404 with addition of 300 µM of acetosyringone for 45 min, two days of pre-culture and three days of co-cultivation periods were determined to be ideal for J. curcas callus transformation. Putative transformants were selected in the presence of 25 mg/l hygromycin. Surviving calli were transferred into proliferation media (MS with 1 mg/l NAA and 1 mg/l BAP) to proliferate the callus for further molecular analyses and to confirm the presence of the target GFP transgene in the putative transformants. Polymerase chain reaction (PCR) was carried out using a 35S specific primer pair confirmed the presence of the 454 bp of 35S promoter region from the transformed callus. Quantitative real-time PCR (qRT-PCR) was carried out to demonstrate the integration and copy number of the 35S promoter in the putative tranformants. The 35S promoter gene (178 bp) as a target gene and J. curcas actin gene (179 bp) which functions as reference gene was designed to detect the positive transformants and control sample in real-time PCR reaction analysis. The results indicated that the actin specific PCR product was present in both the control and transformed calli, however the 35S PCR product was found only in the positive transformants. The similarity in CT values confirmed that both the genes were present as single copy thus confirming a single integration event.

Aspergillus flavusand Aflatoxin Contamination of Poultry Feeds in Tamil Nadu, India

A survey was conducted during 2013 in different regions of Tamil Nadu, India and a total of 51 poultry feed samples were collected from poultry farms and poultry feed dealers. The presence of Aspergillus flavus in the feeds was detected by a polymerase chain reaction (PCR) assay using specific primers based on the O-methyltransferase gene (omt-A) that is involved in the aflatoxin B1 biosynthesis. The contamination levels of Aflatoxin (AFB1, AFB2, AFG1 and AFG2) were determined using high-performance liquid chromatography (HPLC). The results indicated that 98% of the tested samples were contaminated with AFB1 and the levels ranged from 0 to 160.7 ppb and the levels of AFB1 in 29% of the samples exceeded 20 ppb. Aflatoxin B2 contamination was observed in more than 82% of the samples tested and the levels of AFB2 in 17% of the samples exceeded 20 ppb. The levels of AFB2 ranged from 0 to 95.5 ppb. AFG1 and AFG2 were not detected in any of these samples. In the PCR analysis using omt-A primers, A. flavus specific 300-bp PCR product was amplified from DNA extracted from most of the AFB1 contaminated feeds. However, some of the feed samples even though contain AFB1 failed to amplify the 300-bp product in PCR.

Effects of Upconversion Nanoparticles on Polymerase Chain Reaction

Nanoparticles (NPs) are attractive materials owing to their physical and electrochemical properties, which make them extremely useful in diagnostic applications. Photon upconversion is the phenomenon where high-energy photons are emitted upon excitation of low-energy photons. Nucleic acids detection based on upconversion nanoparticles (UCNPs), which display a high signal-to-noise ratio and no photobleaching, has been widely applied. We evaluated whether UCNPs can improve polymerase chain reaction (PCR) specificity and affect PCR amplification. The effects of UCNPs with a diameter size of 40, 70, and 250 nm were evaluated using 3 PCR kits (AccuPower PCR PreMix, AmpliTaq Gold 360 Master Mix, and HotStarTaq Plus Master Mix) and 3 real-time PCR kits (AccuPower GreenStar qPCR PreMix, SYBR Green PCR Master Mix, and QuantiTect SYBR Green PCR Kit). Quantum dots were used for comparison with the UCNPs. In the presence of an appropriate concentration of UCNPs, PCR specificity was optimized. UCNPs of 40-nm size improved PCR specificity more effectively than did UCNPs sized 70 or 250 nm. As the size and concentrations of the UCNPs were increased, PCR amplification was more severely inhibited. At lower annealing temperatures (25°C–45°C), addition of the 40 nm UCNP (1 µg/µL) to the PCR reagent produced specific PCR products without nonspecific sequence amplification. Therefore, UCNPs of different sizes, with different DNA polymerases used in the commercial kits, showed different inhibitory effects on PCR amplification. These results demonstrate that optimization of UCNPs, added to reaction mixtures at appropriate concentrations, can improve PCR specificity. However, the mechanism underlining UCNPs effect on PCR remains unclear and will require further investigation.

Influence of Cytotoxic T-Lymphocyte Antigen-4 Polymorphisms on Acute Rejection Onset of Cadaveric Renal Transplants

We retrospectively examined in cadaveric renal transplants the association between acute rejection episodes (ARE) and single nucleotide polymorphisms (SNPs) localized in the cytotoxic T-lymphocyte antigen (CTLA)-4 promoter, −1147T/C and −318C/T, in exon 1 +49A/G and within the 3′ untranslated region (UTR) CT60G/A. Each one of these SNPs may influence the cell surface expression of the CTLA-4 molecule. Seventy-two cadaveric renal transplant recipients with at least 6 month's follow-up were genotyped for CTLA-4 dimorphisms using direct sequencing of specific polymerase chain reaction products. Allele frequencies in both groups of patients with or without acute rejection (ARE and non-ARE) did not show significant differences in various nucleotide positions. At the level of genotype frequency we first noted a positive association to acute rejection of G/G genotypes (ARE af = 14.7%, non-ARE af = 5.9%) for the +49 (cod. 17), which was associated with decreased expression of the CTLA-4 full-length molecule. In contrast, the AG genotype seemed to be protective (61.8% vs 32.4%, P = .028; odds ratio [OR] = 0.2961). Regarding the CT60G/A dimorphism, noteworthy was the identification of a significantly higher incidence of CT60 A/A genotype in ARE compared with non-ARE group (29.7% vs 8.6%; Yates P = .035; OR = 4.51). Such association of protective AA genotype with ARE, as observed also in autoimmunity, was associated with an increased level of sCTLA-4 induced by the polymorphism, which blocks B7–flCTLA-4 interactions, enhancing T-cell reactivity by preventing transduction of inhibitory signals. Considering the various polymorphic sites in the haplotype, we observed a significant increase in ARE among patients of the CTLA4 +49A/CT60A (HF = 51.5% vs 29.5%; P = .014; OR = 2.545) and a reduction among the +49A/CT60G (17.6% vs 33.8%; P = .04; OR = 0.4193) 2-loci haplotype, As regards the −1147/−318/+49/CT60 CTLA-4 4-loci haplotypes, we observed a significantly higher frequency of the CCAA haplotype in ARE patients comparison with those free of rejection (HF = 51.8% vs 31.1%, P = .046 OR = 2.363). These findings are consistent with those observed in allogeneic stem cell transplantation, wherein patients with CT60 AA showed a major incidence of graft-versus-host disease. An association of protective AA genotype with ARE, as observed also in autoimmunity was associated with an increased level of sCTLA-4 induced by this polymorphism, which blocking the B7–flCTLA-4 interaction, would enhance T-cell reactivity by preventing transduction of inhibitory signals.

Canine-Specific PCR Assay Targeting Cytochrome b Gene for the Detection of Dog Meat Adulteration in Commercial Frankfurters

This report described a cytochrome b (cytb)-based polymerase chain reaction (PCR) assay for the detection of canine tissues in commercial frankfurters. Discriminating detection of canine derivatives in processed food products has important application in halal authentication as well as in health, religions, and fare trades. The assay based on a pair of canine-specific primers that targeted a 100 bp region of canine mithochondrial-cytb gene which is present in multiple copies and highly conserved within the same species. The specificity of the assay was tested against dog and eight most common animal meat species as well as five plant species commonly found in frankfurter formulation. The stability and specificity of the assay were verified under different thermal processing conditions under pure and complex matrices. Three commercial brands of chicken and beef frankfurters were tested in triplicate, and specific PCR products were obtained only from deliberately contaminated formulations. The detection limit of the assay was 0.1 % (0.02 ng DNA) of canine meat spiked with other meats in a typical frankfurter formulation. Shorter amplicon length, superior stability, and higher sensitivity of the assay suggested its potential application in the screening of canine-origin biomaterials in processed food products.