Touchdown PCR Research Articles

Identification of a novel SNP mutation causing drop-out alleles in a paternity test using combined nest and touch-down PCR with Sanger sequencing

Background Short tandem repeat (STR) markers are widely used in forensic DNA analysis due to their ability to provide automated and standardised typing. However, incorrect STR typing can have a significant impact on forensic outcomes. Aim In this study, we detected drop-out alleles at the SE33 locus in a putative father-son pair using the Microreader™ 28 A ID System. This result could lead to a false conclusion of non-paternity. Subjects and methods To investigate the cause of the drop-out alleles, we developed a nest and touch-down PCR program for Sanger sequencing of the SE33 locus. Subsequently, we investigated the mutation frequency in 300 unrelated individuals and reviewed the results of 429 paternity tests. Results The results showed that the frequency of the G > T mutation at this locus was less than 0.01, which is a novel and rare mutation. Our analysis revealed a novel G > T mutation in the primer-binding region of both samples, which was a rare single-nucleotide mutation site in the Chinese population. This variation was found to be responsible for the drop-out alleles observed in the samples. Conclusion Our findings have important implications for optimising primer design and constructing DNA databases for forensic analysis.

Preliminary study of molecular identification of Mycobacterium bovis from cow’s milk in Lorestan (Iran)

Bovine tuberculosis is one of the most important common infectious diseases between humans and livestock. Cow’s milk can be investigated as one of the transmission reservoirs of the disease. Our study was conducted to investigate the presence of Mycobacterium bovis (M. bovis) DNA in cow’s milk from different regions of Lorestan province using Touch-down PCR (TD-PCR) method. So, 100 milk samples from industrial and traditional cattle farms were collected and evaluated according to the animal breed, the average age of the animal and the region where the animal was kept. Seven (26.9%) out of the 26 cow’s milk samples contaminated with Mycobacterium spp., were diagnosed as M. bovis positive. The cattle with an average age of more than 5 years were most infected with Mycobacterium. Also, the crossbred cattle with an average age of 3 to 5 years, which were kept and raised in tropical areas, showed the highest rate of M. bovis infection. Based on our knowledge, this is the first study regarding the presence of Mycobacterium in cow’s milk in Iran.

Detection Of CCR5 Delta-32 Mutation Using High-Resolution Melting Curve Analysis: Challenges and Facts.

The C-C chemokine receptor type 5 (CCR5) is a major co-receptor for human immunodeficiency virus (HIV). Some individuals carry the CCR5 delta-32 genetic polymorphism. People with homozygous CCR5 delta-32 gene are nearly completely resistant to HIV-1 infection. High-resolution melting curve (HRM) analysis is a post-PCR technique utilized for identifying genetic variations in a quick, affordable, and closed-tube assay. The objective of this study was to develop an HRM assay for easy detection of delta-32 mutations. DNA was extracted from peripheral blood mononuclear cells. HRM was performed to detect delta-32 mutation. The study investigated the impact of various factors, including annealing temperature, template concentration, touchdown PCR, additives, amplicon size, and program settings, on HRM Tm differentiation. It was expected that there would be a 4°C Tm difference between amplicons with and without delta-32 mutation, but the test showed a difference of only 2.3°C. In attempts to identify heterozygote delta-32 variants, a Tm difference of only 0.4°C could be achieved. Various modifications were applied, such as adjusting the template concentration, using touchdown PCR, and adding DMSO and glycerol. However, none of these changes helped to differentiate the Tm effectively, especially in delta-32 heterozygote samples. The HRM test identified four samples with heterozygote mutations in each HIV-infected (8.89%) and control (5.72%) groups. More importantly, this study showed that identifying the delta-32 mutation of the CCR5 gene using HRM assay is not as straightforward as previously suggested in some literature, and it requires special setup conditions.

Studying the Pathogenic Effect of New Nucleotide Changes in the Promoter Region of the TERT Gene in Patients with Glioblastoma Brain Tumor

Introduction: Brain tumors are considered to be one of the most dangerous types of cancer, despite decades of research and treatment efforts. The activation of the telomerase enzyme is a critical factor in the immortality of these cells. Mutations in the promoter region of the TERT gene, particularly in the promoter hot spots, can influence the binding of activating transcription factors and inhibit the binding of negative regulatory factors of the TERT gene, ultimately regulating the gene's expression. This study aimed to identify and investigate the mutations of the TERT gene promoter region in glioblastoma, a malignant brain tumor, using bioinformatics. Methods: A study was conducted using the case-control method where 35 patients with glioblastoma multiform brain tumor were examined along with 40 people who were examined as control samples. In this research, the Touchdown PCR technique and direct DNA sequencing were used to detect mutations in the promoter region of the TERT gene in patients with glioblastoma. Furthermore, bioinformatic analyses were conducted to investigate the pathogenic effect of nucleotide changes in this gene region. Results: In the core region of the TERT gene promoter, three-point mutations were identified (c.*146C>T، c.*144C>G and c.*143G>C). Two of these mutations were novel and have been observed for the first time in glioblastoma multiform. The remaining mutation was located in the promoter core's hot spot of the gene. This mutation was known as c.*146C>T. Conclusion: In this research, the harmful impact of TERT promoter region mutations as a biomarker for glioblastoma was examined. These findings suggest that mutations in regulatory regions, as well as coding sequences, could be significant contributory factors in the process of carcinogenesis.

Novel and Sensitive Touchdown Polymerase Chain Reaction Assays for the Detection of Goat and Sheep Milk Adulteration with Cow Milk.

Milk is the most consumed liquid food in the world due to its high nutritional value and relatively low cost, characteristics that make it vulnerable to adulteration. One of the most common types of milk adulteration involves the undeclared addition of cow's milk to milk from other mammalian species, such as goats, sheep, buffalo or donkeys. The incidence of such adulteration not only causes a crisis in terms of commercial market and consumer uncertainty but also poses a risk to public health, as allergies can be triggered by proteins in undeclared cow's milk. In this study, a specific qualitative touchdown (TD) PCR method was developed to detect the undeclared addition of cow's milk in goat and sheep milk based on the discrimination of the peak areas of the melting curves after the modification of bovine-specific primers. The developed methodology has high specificity for the DNA templates of other species, such as buffalos and donkeys, and is able to identify the presence of cow's milk down to 1%. Repeatability was tested at low bovine concentrations of 5% and 1% and resulted in %RSD values of 1.53-2.04 for the goat-cow assay and 2.49-7.16 for the sheep-cow assay, respectively. The application of this method to commercial goat milk samples indicated a high percentage of noncompliance in terms of labeling (50%), while a comparison of the results to rapid immunochromatographic and ELISA kits validated the excellent sensitivity and applicability of the proposed PCR methodology that was able to trace more adulterated samples. The developed assays offer the advantage of multiple detection in a single run, resulting in a cost- and time-efficient method. Future studies will focus on the applicability of these assays in dairy products such as cheese and yogurt.

Primer Design and Amplification of IgM Antibody Repertoire of Philippine Carabao (Bubalus kerabau Fitzinger, 1860) for Construction of Phage Display Library

Antibody display library is a robust platform for the production of recombinant or monoclonal antibodies that are of great value in diagnostics and therapeutics. A naïve library constructed based on immunoglobulin M (IgM) can reflect the natural immunity of the animal and can offer broad antigen targets. As a natural host of various zoonotic pathogens, the Philippine carabao (Bubalus kerabau Fitzinger, 1860) offers great potential as a surrogate model for molecular biology and biomedical applications. However, studies on its antibody repertoire are very scarce, if not available. Hence, this study was undertaken to design and evaluate degenerate primers targeting the antigen-binding paratope of B. kerabau Fitzinger, 1860 IgM – specifically, lambda variable light chain (VLL), kappa variable light chain (VLK), and variable heavy chain (VH). These primers were mainly based on carabao’s close relative in the Bovidae family, the cattle (Bos taurus). A total of 32 degenerate primers, 14 designed and 18 adapted from literature, were used to amplify the target antibody fragments using a high-fidelity DNA polymerase with a modified touchdown–polymerase chain reaction method. Results demonstrate the amplification of VLL, VLK, and VH genes derived from B-cells of carabao splenic tissues using the degenerate primers. The amplicons will be used in the construction of an antibody phage library to elucidate the IgM repertoire of the Philippine carabao. In addition, DNA sequencing surprisingly revealed longer VLL and VLK genes than VH genes in contrast to what is found in other bovines, suggesting a novel organization of immunoglobulin genes of B. kerabau Fitzinger, 1860 that merits further investigation.

Development of a touchdown droplet digital PCR assay for the detection and quantitation of human papillomavirus 16 and 18 from self-collected anal samples

ABSTRACT Broad-spectrum droplet digital polymerase chain reaction (ddPCR) assays can be used to quantitate multiple human papillomavirus (HPV) types. However, the presence of nucleotide mismatches at the primer binding sites may result in different optimal annealing temperatures and, consequently, under detection of some HPV types. Touchdown PCR uses decreasing annealing temperature over the first PCR cycles allowing for specific and efficient amplification of the target sequence. Here, as a proof-of-concept, we assessed if touchdown ddPCR (TD-ddPCR) using broad-spectrum oligonucleotides and genotype-specific probes targeting HPV L1 can be used to quantitate HPV. We tested our hypothesis using HPV positive cell lines and anal samples which potentially contain PCR inhibitors. Using serial dilutions of HPV16 and HPV18 DNA, the TD-ddPCR assays were performed with linearity across the dilution series (Pearson’s correlation r = 0.9999) and detected one copy of both HPV types, with no cross-reactivity to 27 other low-risk and high-risk HPV types. The PCRs were highly reproducible, with inter- and intra-assay coefficient of variations ranging 2.0–6.3% for HPV16 and 4.9–11.6% for HPV18. Testing self-collected anal samples positive for HPV16 ( n = 29) or HPV18 ( n = 33) and those negative for both HPV types ( n = 22) with the ddPCR assay and Seegene Anyplex II HPV28 (Seegene, TD-ddPCR) showed high agreements between the two assays. In conclusion, HPV broad-spectrum primers containing mismatches at their target binding sites and a touchdown thermal cycling approach resulted in sensitive and specific amplification of HPV genome using ddPCR. The assay has potential clinical diagnostic applications for detection, quantitation, and monitoring of anal HPV infections. IMPORTANCE The quantity of the human papillomavirus (HPV) is associated with disease outcome. We designed an accurate and precise digital PCR assay for quantitating HPV in anal samples, a sample type that is typically problematic due to the presence of PCR inhibitors.

Effects of primers, PCR approaches and sample preservation methods on diversity studies of myxobacteria

Myxobacteria have potential application value in developing new antibiotics and environmental protection. In this study, in order to establish a more suitable method for diversity studies of myxobacteria, the effects of primers, polymerase chain reaction (PCR) approaches and sample preservation methods on the results were compared by Illumina high-throughput sequencing. The results showed that the relative abundance and operational taxonomic unit (OTU) ratio of myxobacteria amplified by the universal primers accounted for 0.91–1.85% and 2.82–4.10% of total bacteria, indicating that myxobacteria were the dominant bacteria both in population and species numbers. The relative abundance and OTU number and ratio of myxobacteria amplified by the myxobacteria semi-specific primers were significantly higher than those amplified by the universal primers, of which the primer pair W2/802R specifically amplified myxobacteria of suborder Cystobacterineae, while the primer pair W5/802R mainly amplified myxobacteria of suborder Sorangineae and also amplified more species of suborder Nannocystineae at the same time. Among three PCR approaches, the relative abundance and OTU ratio of myxobacteria amplified by the touch-down PCR were the highest. More myxobacterial OTUs were detected in most dried samples. In conclusion, the combination of the myxobacteria semi-specific primer pairs W2/802R and W5/802R, touch-down PCR, and dry preservation of samples were more conducive to diversity studies of myxobacteria.

Three Previously Unseen Genotypes Detected in IGFBP - 3 Gene in Buffalo Breed

Anatolian water buffalo breed is Turkey’s sole water buffalo breed, and their numbers steadily increased with the national “Water Buffalo Breeding by Breeders Project”. This study aimed to investigate of the gene region polymorphisms (Intron-2, Exon-2/Intron-3, Exon-3) of the meat-yield-related Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) gene in Anatolian water buffaloes by TaqI, HaeIII, and MspI restriction endonucleases. The phenol/chloroform method was used for DNA isolation from 151 blood samples, and extracted DNAs were amplified by touchdown PCR using specific primers. Amplified PCR products were digested with restriction endonucleases (REs) and separated in 3% agarose gel electrophoresis (AGE), then genotypes were determined. Results revealed two genotypes [AA (98.68%) and AC (1.32%)] and two alleles [A (0.99) and C (0.01)] for the Exon-2 to Intron-3 region from HaeIII digestion. TaqI digestion of the Intron-2 region revealed three genotypes [AA (7.94%), AB (3.97%), and BB (88.10%)] and two alleles [A (0.10) and B (0.90)]. MspI digestion of the Exon-3 region revealed only the AA genotype and A allele, thus revealing monomorphism. Overall, HaeIII digestion revealed insignificant polymorphism (P>0.05), and TaqI digestion revealed significant polymorphism (P<0.001) for their respective regions. Gene polymorphisms of these regions were investigated for the first time in Anatolian water buffaloes. Additionally, three novel genotypes for the IGFBP-3 gene (one from HaeIII and two from TaqI) were determined for the first time. The novel B allele from TaqI digestion was observed to have a substantial frequency.

Substantiation of a clopidogrel metabolism-associated gene (CYP2C19) variation among healthy individuals

BackgroundIt is essential to investigate the prevalence of CYP2C19 alleles that affect drug metabolism. This study measures the allelic and genotypic frequencies of CYP2C19 loss-of-function (LoF) alleles CYP2C19∗2, CYP2C19∗3, and gain-of-function (GoF) alleles CYP2C19∗17 in the general population. MethodologyThe study involved 300 healthy subjects between the ages of 18 and 85 recruited by simple random sampling. Allele-specific touchdown PCR was employed to identify the various alleles. The genotype and allele frequencies were calculated and checked for Hardy–Weinberg equilibrium. The phenotypic prediction of ultra-rapid metabolizer (UM = ∗17/∗17), extensive metabolizer (EM = ∗1/∗17, ∗1/∗1), intermediate metabolizer (IM = ∗1/∗2, ∗1/∗3, ∗2/∗17) and poor metabolizer (PM = ∗2/∗2, ∗2/∗3, ∗3/∗3) was made based on their genotype. ResultsThe allele frequency of CYP2C19∗2, CYP2C19∗3, and CYP2C19∗17 was 0.365, 0.0033, and 0.18, respectively. The IM phenotype predominated with an overall frequency of 46.67%, including 101 subjects with ∗1/∗2, two subjects with ∗1/∗3, and 37 subjects with ∗2/∗17 genotype. This was followed by EM phenotype with an overall frequency of 35%, including 35 subjects with ∗1/∗17 and 70 subjects with ∗1/∗1 genotype. PM phenotype had an overall frequency of 12.67%, including 38 subjects with ∗2/∗2 genotype, and UM phenotype had an overall frequency of 5.67%, including 17 subjects with ∗17/∗17 genotype. ConclusionGiven the high allelic frequency of PM in the study population, a pre-treatment test to identify the individual's genotype may be recommended to decide the dosage, monitor the drug response, and avoid adverse drug reactions.

Development of subfamily-based consensus PCR assays for the detection of human and animal herpesviruses

Consensus PCR assays that can be used to sensitively detect several herpesvirus (HV) species across the different subfamilies were developed in this study. Primers containing degenerate bases were designed to amplify regions of the DNA polymerase (DPOL) gene of alpha- and gamma-HVs, and the glycoprotein B (gB) gene of beta-HVs in a singleplex, non-nested touchdown PCR format. The singleplex touchdown consensus PCR (STC-PCR) was used to amplify the DNA of eight human and 24 animal HVs. The assay was able to detect the lowest DNA dilution of 10−5 for alpha-HVs and 10−3 for beta- and gamma-HVs. In comparison, lowest detection limits of 10−5, 10−3, and 10−2 were obtained for alpha-, beta-, and gamma-HVs respectively when a nested PCR was used. The findings in this study suggest that the STC-PCR assays can be employed for the molecular surveys and clinical detection of novel and known HVs.

Multiplex touchdown Polymerase Chain Reaction for rapid detection of Salmonella enterica subsp. enterica serovars Enteritidis and Typhimurium in food

The Salmonella outbreak is one of the leading foodborne diseases in the world with increasing cases being reported annually. However, the current methods for Salmonella detection in foods are outdated, laborious and time-consuming. This necessitated developing a technique that is rapid for Salmonella detection in foods. Thus, the current study aimed to develop a multiplex touchdown PCR (m-TdPCR) protocol for rapid and simultaneous detection of Salmonella enterica subsp. enterica serovars Enteritidis and Typhimurium in foods. A two-phase m-TdPCR protocol was developed and optimized with primer pairs targeting the Salmonella enterica subsp. enterica (ST11/ST15-0.15 µM), serovars Enteritidis (sdfI gene-1.2 µM), Typhimurium (fliC gene-1.5 µM) and an internal amplification control (16S rRNA-0.08 µM). It was found that the m-TdPCR protocol is highly sensitive detecting up to 1 ng of Salmonella DNA and its specificity was verified using the in-silico method. Furthermore, the developed m-TdPCR shows no non-specific PCR amplicons and is able to detect both S. enterica ser. Enteritidis and S. enterica ser. Typhimurium in real-time when tested against the artificially contaminated food samples at up to 10-3 dilutions. Therefore, the validated m-TdPCR protocol in this study can be used as a tool for rapid detection of S. enterica ser. Enteritidis and S. enterica ser. Typhimurium in food samples and this may significantly reduce any related foodborne incidences in future.

The triterpenoid saponin content difference is associated with the two type oxidosqualene cyclase gene copy numbers of Pulsatilla chinensis and Pulsatilla cernua.

Pulsatilla chinensis is an important medicinal herb, its dried radix is used to treat the inflammation since ancient China. Triterpenoid saponins are proved to be the main active compounds of Pulsatilla genus. The triterpenoid saponin contents vary widely in different Pulsatilla species. But no enzyme involved in the triterpenoid saponin biosynthetic pathway was identified in Pulsitilla genus. This seriously limits the explanation of the triterpene content difference of Pulsatilla species. In this article, we obtained two oxidosqualene cyclase (OSC) genes from P. chinensis and P. cernua by touchdown PCR and anchored PCR. These two OSCs converted 2,3-oxidosqualene into different triterpenoids. The OSC from P. cernua is a monofunctional enzyme for β-amyrin synthesis, while the OSC from P. chinensis is a multifunctional enzyme for lupeol and β-amyrin synthesis, and the lupeol is the main product. Then we identified the 260th amino acid residue was the key site for the product difference by gene fusion and site-directed mutant technology. When the 260th amino acid residue was tryptophan (W260) and phenylalanine (F260), the main catalysate was β-amyrin and lupeol, respectively. Then we found that the expression of these two genes was strongly correlated with the lupeol-type and β-amyrin-type triterpenoid contents in P. cernua and P. chinensis. Finally, we found the gene copy number difference of these two genotypes leaded to the triterpenoid diversity in P. cernua and P. chinensis. This study provides useful information for the molecular breeding and quality improvement of P. chinensis and a molecular marker to identify the P. chinensis decoction pieces.

Splice site m6A methylation prevents binding of DGCR8 to suppress KRT4 pre-mRNA splicing in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the 11th most prevalent tumor worldwide. Despite advantages of therapeutic approaches, the 5-year survival rate of patients with OSCC is less than 50%. It is urgent to elucidate mechanisms underlying OSCC progression for developing novel treatment strategies. Our recent study has revealed that Keratin 4 (KRT4) suppresses OSCC development, which is downregulated in OSCC. Nevertheless, the mechanism downregulating KRT4 in OSCC remains unknown. In this study, touchdown PCR was utilized to detect KRT4 pre-mRNA splicing, while m6A RNA methylation was identified by methylated RNA immunoprecipitation (MeRIP). Besides, RNA immunoprecipitation (RIP) was used to determine RNA-protein interaction. Herein, this study indicated that intron splicing of KRT4 pre-mRNA was suppressed in OSCC. Mechanistically, m6A methylation of exon-intron boundaries prevented intron splicing of KRT4 pre-mRNA in OSCC. Besides, m6A methylation suppressed the binding of splice factor DGCR8 microprocessor complex subunit (DGCR8) to exon-intron boundaries in KRT4 pre-mRNA to prohibit intron splicing of KRT4 pre-mRNA in OSCC. These findings revealed the mechanism downregulating KRT4 in OSCC and provided potential therapeutic targets for OSCC.

Comparative analysis of A1 and A2 allele detection efficiency for bovine CSN2 gene by AS-PCR methods.

This study was aimed at conducting a comparative analysis of the efficiency in genotyping cattle by beta-casein locus using the allele-specific PCR methods. The results of the study have demonstrated the necessity to optimize the protocol for the use of AS-PCR to detect alleles A1 and A2. It was found that the use of non-optimized PCR protocols led to the genotyping errors, manifested regarding beta-casein locus (CSN2). The impossibility of using the touchdown PCR as an optimization instrument for AS-PCR was proven. The elaborated typing protocols were used to study the genetic structure of the cattle populations of different breeds, reared in Ukraine - Ukrainian Red-and-White dairy, Ukrainian Black-and-White dairy (two populations), and Charolais. It was found that locus CSN2 was polymorphic in all the cattle populations. The frequencies of allele A2 varied within 0.34-0.91 depending on the population of the animals, which may be conditioned by the specificities in the selection work. No deviation from the Hardy-Weinberg equilibrium was found in any investigated population of cattle.

Requiring Reconsideration of Differences of Aeromonas Infections Between Extra-Intestinal and Intestinal in Hospitalized Patients.

The purpose of this study is to examine the variations between extra-intestinal and intestinal infections of Aeromonas in terms of strain types, risk factors, drug susceptibility results, and the distribution of drug resistance and virulence genes. A total of 188 Aeromonas strains were identified to the species level using housekeeping genes (rpoD, gyrB, and gyrA). The risk factors for Aeromonas extra-intestinal and intestinal infection, as well as mortality, were retrospectively examined in this study. The broth microdilution method was used to investigate the antimicrobial susceptibility profiles. Touchdown polymerase chain reaction (PCR) assays and DNA sequencing were employed to confirm virulence and the presence of drug resistance genes. The housekeeping genes identified 188 strains into 7 species. Extra-intestinal isolates generally contained A. caviae and A. hydrophila, while intestinal were A. veronii (p=0.0001). Extra-intestinal infections (158/188) were the main type and accounted for 24/27 of all fatalities. Malignant tumors, hepatobiliary diseases, anemia, and hypoproteinemia were linked to infections. Poor results were associated with septic shock. Using the broth microdilution method, over 80% isolates were susceptible to most antimicrobials, except for ceftazidime (79.8%) and ceftriaxone (69.7%). Except for imipenem, intestinal strains were more susceptible to other medications than extra-intestinal. Using touch-down polymerase chain reaction testing and DNA sequencing, 6 strains, 31 strains, and a strain only had bla TEM, bla CphA, and bla VIM, respectively. Two Aeromonas hydrophila each possessed bla CphA+ bla CTXM-M-9, and bla CphA + bla CTX-M-1 + bla CTX-M-15-like + bla TEM; two Aeromonas caviae each possessed bla NDM + bla CTX-M-1 +bla CTX-M-15-like + bla TEM, and bla NDM + bla TEM. Thirty-four of the 42 strains mentioned above were isolated from extra-intestinal. Act, aexT, and ascF-G, were in intestinal more frequently, but alt, hlyA, ela, and lip were in extra-intestinal more frequently. Aeromonas inside and outside intestinal differed in their clinical characteristics, drug susceptibility, drug resistance and virulence genes.

Mastadenovirus Molecular Diversity in Waste and Environmental Waters from the Lisbon Metropolitan Area.

In face of the absence of epidemiological data regarding the circulation of human adenoviruses (HAdV) in Portugal, this study aimed at the evaluation of their molecular diversity in waste and environmental waters in the Lisbon Metropolitan Area (LMA). Using samples collected between 2018 and 2021, the HAdV hexon protein-coding sequence was partially amplified using three nested touch-down PCR protocols. The amplification products obtained were analyzed in parallel by two approaches: molecular cloning followed by Sanger sequencing and Next-Generation Sequencing (NGS) using Illumina® sequencing. The analysis of NGS-generated data allowed the identification of a higher diversity of HAdV-A (19%), -B (1%), -C (3%), -D (24%), and -F (25%) viral types, along with murine adenovirus (MAdV-2; 30%) in the wastewater treatment plant samples. On the other hand, HAdV-A (19%), -D (32%), and -F (36%) were identified in environmental samples, and possibly MAdV-2 (14%). These results demonstrate the presence of fecal contamination in environmental waters and the assessment of the diversity of this virus provides important information regarding the distribution of HAdV in LMA, including the detection of HAdV-F41, the most frequently reported in water worldwide.

Mutant Allele of CD44 (rs8193C>T) and Pum2 Regulatory Element as A Prognosis Factor of Prostate Neoplasms: A Case-Control and In Silico Studies.

Expression of CD44 variant 6 (CD44v6) as a homing-associated cell adhesion molecule (HCAM), has proved to change most cancer cells. Aim of the study is the effect of mutant allele of CD44 (rs8193C>T) and Pum2 regulatory element as a prognosis factor of prostate neoplasms: a case-control and in silico studies in the Mazandaran province-Iran. In a case-control study, CD44-rs8193C>T genotyping of the 420 prostate neoplasms (210 benign prostatic hyperplasia (BPH) patients and 210 prostate cancer patients) and 150 healthy samples are performed by the touchdown polymerase chain reaction with confronting two-pair primers (PCR-CTPP) method. The T mutant allele effects on the mRNA structure and cell pathways were also investigated in silico methods. Our results showed that the increase of T mutant allele frequency was significantly associated with BPH compared with prostate cancer. Furthermore, results showed TT genotype was significantly associated with BPH [odds ratio (OR)=0.572 and P=0.015], and also influenced the CD44v6 transcript secondary structure, miRNA binding, and regulatory element-binding site for Pum2 protein. Attachment of Pum2 to standard CD44 transcript may lead to transcript isoform-switching and shift-expression to a variety of CD44 isoforms, which can trigger some of the cell signaling pathways, such as Nanog-Stat, PKC-Nanog, and PKC-Twist. Based on this, the presence of the T mutant allele of CD44 (rs8193C>T) in the populations may create a regulatory element-binding site for Pum2. So, it could be known as a prognosis factor and prediction of prostate neoplasms. However, more comprehensive studies in different populations (with various ethnicities and large population sizes), and also CD44v6 gene expression studies in protein and transcript levels are required to confirm our data.

TOUCHDOWN PCR FOR INCREASED SENSITIVITY AND SPECIFICITY IN AMPLIFICATION OF MOUSE COGNATE HOMEOBOX

Mammalian system have a large genome with a high level of gene sequence identity from other genomic DNA, making assessment difficult and time-consuming.Our findings describe a simple method for rapidly isolating and amplifying HoxA loci in the mouse genome using degenerate primers. For the semi degenerate primers,they were designed based on cognate gene coding regions of consensus sequences. After assembling sequences from different primer matches amplifying the same HoxA loci, the effects of the universal primer-template match on the efficiency of standard PCR amplification were investigated. Touchdown PCR increased specificity and yield in two consecutive amplifications on different gel concentrations by using high and low annealing temperatures. This method was quick, simple, and inexpensive for amplification of consensus sequences in very large gene sequences.

Short Communication: Prevalence of Listeria monocytogenes in Raw Milk of Healthy Sheep and Goats.

Listeria monocytogenes, one of the most important bacterial pathogens transmitted through milk, causes listeriosis in humans and animals. This study aimed to investigate the prevalence of L. monocytogenes in raw milk of healthy sheep and goats in the west of Iran (Lorestan Province) by touchdown PCR (TD-PCR). Listeria spp. were found in milk samples taken from 21 sheep (29.16%) and 3 goats (10.71%) whereas L. monocytogenes was isolated from milk samples taken from 4 sheep (5.55%) and 1 goat (3.75%). The results showed that there was a significant difference between sheep and goats in the prevalence of Listeria spp. in their raw milk (p < 0.05), but no significant difference was observed between them in the prevalence of L. monocytogenes. The study findings suggested that the raw milk of healthy sheep and goats was infected with L. monocytogenes and warned of the risk of human infection with listeriosis following consumption of raw and unpasteurized milk.