Polymerase Chain Reaction Reaction Research Articles

Antimicrobial resistance patterns and virulence determinants of clinical enterococcus isolates in Pakistan

Background. The current study was designed to determine antibiotic resistance profile,detection of antimicrobial resistance and virulence-related genes among enterococcus species. Materials and methods. Altogether, one hundred fifty enterococcal isolates were collected from various clinical specimens and identified by Polymerase chain reaction (PCR). Antibiotic susceptibility testing and MICs of vancomycin were carried out as per CLSI guidelines. A series of PCR reactions were used to screen vancomycin-resistant genes (vanA, vanB, and vanD) and virulence-related genes (esp, ace, asa1, gelE cylA) among VRE enterococcus species. Results. The isolated enterococcal strains comprised 62.6% E. faecalis, 33.4% E. faecium, and 4% of other species. Overall enterococcus showed a high level of resistance; 94% to erythromycin, followed by ciprofloxacin 82.6%, levofloxacin 70%, and vancomycin 16%. The 57.4% of the isolates were recovered from hospitalized patients and 96% of the enterococcus isolates were multi-drug resistant. The MICs of vancomycin-resistant strains remained in the range of 32 µg/ml to 256 µg/ml for the majority of the isolates. The vancomycin-resistant phenotypes vanA, vanB, and vanD were found in 29.2%, 37.5%, and 33.3% isolates respectively. Regarding virulence determinants the observed percentages were as follows; esp: 16.6%, asa1: 70.8%, gelE: 25%, ace: 33.3%, and cylA: 25%. Conclusion. The majority of the isolates were E. faecalis and multi-drug resistant. The VRE isolates carried antimicrobial resistance and virulence-related genes, and vanA, B, D phenotypes were the most common among VRE isolates.

A Thermal Cycler Based on Magnetic Induction Heating and Anti-Freezing Water Cooling for Rapid PCR.

Distinguished by its exceptional sensitivity and specificity, Polymerase Chain Reaction (PCR) is a pivotal technology for pathogen detection. However, traditional PCR instruments that employ thermoelectric cooling (TEC) are often constrained by cost, efficiency, and performance variability resulting from the fluctuations in ambient temperature. Here, we present a thermal cycler that utilizes electromagnetic induction heating at 50 kHz and anti-freezing water cooling with a velocity of 0.06 m/s to facilitate rapid heating and cooling of the PCR reaction chamber, significantly enhancing heat transfer efficiency. A multi-physics theoretical heat transfer model, developed using the digital twin approach, enables precise temperature control through advanced algorithms. Experimental results reveal average heating and cooling rates of 14.92 °C/s and 13.39 °C/s, respectively, significantly exceeding those of conventional methods. Compared to commercial PCR instruments, the proposed system further optimizes cost, efficiency, and practicality. Finally, PCR experiments were successfully performed using cDNA (Hepatitis B virus) at various concentrations.

Detection of DNA of Leishmania infantum in the brains of dogs without neurological signs in an endemic region for leishmaniasis in the state of Rio Grande do Sul, Brazil.

Canine visceral leishmaniasis (CVL) is a parasitic disease caused by the protozoan Leishmania infantum. Neurological infection occurs due to the parasite's ability to cross the blood-brain barrier. It is known that dogs can remain infected with a subclinical infection for life, potentially acting as reservoirs for L. infantum when bitten by sandflies. In this context, the objective of this study was to evaluate the occurrence of Leishmania spp. in the brains of dogs from the metropolitan region of Rio Grande do Sul, Brazil, without a history of neurological disease but residing in an endemic area for L. infantum. A total of 200 samples, from 2022 to 2023, were evaluated using conventional Polymerase Chain Reaction (PCR) with the primers Leishmini-F GGKAGGGGCGTTCTGC and Leishmini-R STATWTTACACCAACCCC, aiming to amplify a product of 120 base pairs for Leishmania spp. To identify the species, a multiplex PCR was used, differentiating L. braziliensis (127bp), L. amazonensis (100bp), and L. infantum (60bp), with the molecular target being the conserved region of the kinetoplast DNA (kDNA) minicircle, specific to Leishmania spp. Of the 200 samples evaluated, 26.5% (53/200) tested positive in the conventional PCR reaction for Leishmania spp., with the PCR multiplex the only species detected was Leishmania infantum. The average age of the positive animals was 5.08years, with 47.2% being females and 52.8% being males; among these, mixed-breed dogs were the most predominant, representing 43.4% of the total. Clinical signs varied: hepatomegaly in two dogs, pronounced neutrophilic hepatitis in one, splenomegaly in one with lymphoid hyperplasia, and glomerulonephritis and nephritis in two animals. Mild anemia and thrombocytopenia were found in eight, with pale mucous membranes in three, and diffuse alveolar edema in one case. Notable pathological findings included suspected distemper in one animal and lymphoplasmacytic meningitis in another. Histopathological findings revealed alveolar edema and acute renal failure. A third dog exhibited bilateral hydrocephalus and diffuse edema in the brain. Additional changes, such as mild inflammatory infiltrate and slight vacuolar degeneration, were observed in 11.3% of the analyzed brains. There was no clinical suspicion of leishmaniasis in any of the studied cases. Therefore, the detection of L. infantum DNA in the brains of dogs suggests that animals with subclinical infection may play a crucial role in the spread of leishmaniasis, and infection by Leishmania spp. should be considered as a differential diagnosis for neurological disease in endemic areas for the protozoan.

Molecular prevalence of Borrelia burgdorferi, Ehrlichia canis, and Coxiella burnetii in dogs and associated ticks in Egypt: Emerging One Health challenging zoonoses.

Tick-borne pathogens pose a significant problem in canines, other animals, and humans worldwide. This study aimed to estimate the prevalence of Borrelia burgdorferi, Ehrlichia canis, and Coxiella burnetii in dogs and associated ticks in Egypt. Blood samples from 110 tick-infested dogs and 550 whole ticks (divided into 110 pools) were collected and tested for the targeted pathogens using polymerase chain reaction (PCR). Of the 110 dog blood samples, B. burgdorferi DNA was detected in three samples, E. canis in six samples, and C. burnetii in one kenneled dog. Among the 110 tick pools, B. burgdorferi was detected in four pools, E. canis in 12 pools, and C. burnetii in three pools from kenneled dogs. The overall prevalence of the three agents in dog and tick samples were 3.18%, 8.18%, and 1.81%, respectively. Simultaneous positive PCR reactions in both dogs and their associated tick pools were observed in four cases. B. burgdorferi and E. canis were simultaneously detected in two dogs and two tick pools, whereas C. burnetii was detected in one dog but not in any tick pools. The three agents were simultaneously detected in one dog, but none were found in the corresponding tick pools. A mixed infection of C. burnetii and B. burgdorferi was observed in one dog and one tick pool. Molecular diagnosis is the most reliable method for detecting B. burgdorferi, E. canis, and C. burnetii in dogs and associated ticks. E. canis showed the highest prevalence in both dog and tick samples followed by B. burgdorferi while C. burnetti showed the lowest prevalence. The potential transmission of these diseases from companion dogs to humans through ticks presents a significant challenge for the One Health concept.

A PCR-Based Approach for Early Diagnosis of Head and Neck Aspergillosis: A Pilot Study.

Aspergillosis is a fungal disease caused by the inhalation of fungal spores of the genus Aspergillus spp. This fungus mainly affects the lungs but can spread and infect the maxillofacial region through the bloodstream or inoculation of the fungus after extraction or endodontic treatment, especially in the upper posterior teeth. The disease has nonspecific clinical manifestations that hinder its early diagnosis. Although the Polymerase Chain Reaction (PCR) technique holds promise as a diagnostic tool for aspergillosis, anatomopathological analysis services do not routinely adopt this method. Therefore, the present study aimed to evaluate the applicability of PCR and standardise the techniques of preparation of biological samples for the detection of the three species: Aspergillus niger, Aspergillus fumigatus and Aspergillus flavus. Methods: Six samples of formalin-fixed, paraffin-embedded tissue (FFPE) with a histopathological diagnosis suggestive of aspergillosis were investigated using PCR. As a positive control for the PCR reaction, morphologically and genetically characterized cultures were used, with their sequences deposited at NCBI under accession codes MW837777 (A. fumigatus) and MW837779 (A. niger). The A. flavus culture used is reference RC 2053. Four of the six samples evaluated were positive for Aspergillus spp., of which one was co-infected with A. fumigatus and A. flavus species, while two others were positive only for A. flavus, and one sample was positive only for A. fumigatus. These findings suggest that PCR can be used as an auxiliary method for diagnosing aspergillosis. However, this was a pilot study, and expansion of the sample size and the evaluation of PCR in comparison with other diagnostic tests for aspergillosis are essential to determine the accuracy of the method.

B-208 Room Temperature Stable PCR Reagents Preparation Using Capillary-Mediated Vitrification Process

Abstract Background Polymerase chain reaction (PCR) is a sensitive, and most used molecular technique for the disease diagnosis. Reagents used for PCR is available as a one-step and ready-to-use PCR Master Mix, which contains Taq DNA polymerase, dNTPs, MgC12, PCR reaction buffer. However, to preserve bioactivity, PCR reagents must be stored and distributed as frozen. Desiccation of PCR reagents can avoid cold storage and improper handling of wet reagents and its associated errors. We discovered capillary-mediated vitrification (CMV) processes can stabilize different biomolecules and allow them to be stored at ambient temperature. Here, we hypothesized that the CMV process can preserve and improve PCR reagents thermal stability, which will reduce PCR processing time and improve the testing throughput. Methods The CMV process was performed by mixing PCR Master Mix individual or all reagents such as Taq DNA polymerase or Hot Start Taq DNA polymerase, dNTPs, 16S rRNA primers, MgC12, and PCR reaction buffer with an excipient buffer and adding the mixture to a porous scaffold. The loaded scaffolds were placed into our vitrification chamber and dried for 30 min. After drying, the CMV-stabilized sample was tested or packed in Mylar pouches and were stored at ambient and elevated temperatures for 42 days. On the day of testing, the stabilized samples were eluted with nuclease-free water and E. coli genomic DNA was added, and the 50 uL reaction was tested by conventional PCR. The PCR product was visualized under agarose gel electrophoresis. Results To assess whether the CMV process maintains PCR reagents activity and not interfering with the assay, the individual PCR reagents and as a Master mix are preserved. The CMV stabilized polymerase enzyme, primer, or dNTPs amplified DNA similar to frozen reagents and produced comparable PCR products. Notably, CMV stabilized PCR Master Mix which contains either Taq DNA polymerase or Hot Start Taq DNA polymerase enzyme, dNTPs, primers, MgC12, and reaction buffer yielded intact amplified PCR product, which is comparable to frozen reagents. Likewise, a high intense PCR product was observed in the stabilized PCR Master Mix reagents when stored at room and elevated temperatures for 42 days. Conclusions The study results indicate that the CMV process stabilized and improved the PCR Master Mix reagents activity. The study suggests that the CMV technology has the potential to enable ambient storage and shipping of PCR reagents for molecular diagnostic and other related applications.

KRAS mutations detection methodology: from RFLP to CRISPR/Cas based methods.

In personalized cancer medicine, the identification of KRAS mutations is essential for making treatment decisions and improving patient outcomes. This work presents a comprehensive review of the current approaches for detection of KRAS mutations in different cancers. We highlight the value of fast and reliable KRAS mutations discovery and the effectiveness of molecular testing for selecting individuals who might benefit from targeted therapy. We provide an overview of various methods and tools available for detecting KRAS mutations, such as digital droplet PCR, next-generation sequencing (NGS), and polymerase chain reaction (PCR). We also address the difficulties and limitations in the identification of KRAS mutations, namely tumor heterogeneity and the emergence of resistance mechanisms. This article aims to guide clinicians in KRAS mutation identification.

Molecular Detection of Human parvovirus B19 in Patients with Thalassemia in Wasit Province, Iraq

Background: Human B19 (PVB19) isssmall, non-envelope single strand DNA viruses from family of "Parvoviridae" it cause infections in human. In thalassemia are a heterogeneous grouping of genetic disorders that result from a decreased synthesis of alpha or beta chains of hemoglobin (Hb). Numerous factors contribute to functional abnormality found in βeta thalassemia patient like decrease red cells life span, rapidsiron turnover, and tissue deposition of excess iron B19 targets the erythroid progenitors in the bone marrow by binding to the glycosphingolipid. Materials and Methods: This case cross sectional study and the patients aged between (5-40) years , study has been carried outsin Al-Kut teaching hospitals in Al-Iraq-Wasit in a period between September 2023 and March 2024. This study followed the cross-sectional design. The populations of our study included 120 patients’ samples with thalassemia infected by Human Parvovirus B19 in blood transfusion patients 64 male and 56 female. Nested-PCR used to detect of B19V by using primers. PCR reaction was done by using special sets of primers mentioned previously and special components and specific program and procedure according to components of PCR reaction. Result: Human parvovirus 19 Positive case percentage using polymerase chain reaction indicated that the positive cases was reported according to polymerase chain reaction (PCR) as a percentage of case related to the main characteristic of patients, current study data has been indicated as following: in total of 120 patient sample with thalassemia has been included in this study, PCR positive case was 17 (14.2%), 6 (5%), 2 (1.7%) and 0 (0%) in the age groups (5-15), (16-25), (26-35) and (36-45) respectively. Male was more prevalent 17 (14.2%), than female 8 (6.7%), So as to records of major type in comparision to intermediate 19 (15.8%) and 6 (5%). All positive cases were 25 cases. Conclusion: Current data showed prevalence of Human parvovirus 19 in Iraqi patients. This prevalence should be alarming public health.

NEW PRIMER DESIGN of MULTIPLEX POLYMERASE CHAIN REACTION (PCR) for DETECTION of Escherichia Coli and Salmonella Enterica in FOOD SAMPLE

Background: Multiplex PCR techniques are used to detect multiple pathogens at the same time simultaneously. In order to achieve the specificity and sensitivity of detection, it is important to optimize the multiplex PCR method properly. Developing suitable primers and optimizing PCR temperature to boost particular genes from pathogens are key factors for this optimization. Methods: For the simultaneous detection of Escherichia coli and Salmonella enterica in food samples, this research aims to develop a set of primers for multiplex polymerase chain reaction (PCR). Using Primer-BLAST software, the study utilizes specific primer designs for the phoA gene (Eschericia coli) and invA gene (Salmonella enterica). DNA isolation has confirmed successful extraction from the two bacterial samples. PCR was performed under different conditions, including Singleplex and Multiplex PCR, using two annealing temperatures of 53oC and 50oC. Results: The results showed that this method can effectively amplify target genes and indicate their specificity and reliability at both temperature levels. Given these results, it has successfully conducted multiplex PCR using the built primer pairs. Both annealing temperatures of 50oC and 53oC can be used to perform multiplex PCR to detect E. coli and Salmonella enterica in one PCR reaction. Conclusion: Through this research, we have created a new set of Multiplex PCR Primers and an optimized multiplex PCR technique for the simultaneous detection of E coli and Salmonella enterica in food samples. The rapid and simultaneous screening of E. coli and S. enterica, which contributes to improved food safety measures and pathogen detection in the food industry, is promising with this optimized PCR approach.

Development of a multiplex real-time quantitative reverse-transcription polymerase chain reaction for the detection of four bee viruses

Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.

Thermally resistant nuclease in Serratia marcescens hinders PCR reactions and degrades PCR products.

Polymerase chain reaction (PCR) is an important tool for exogenous gene acquisition and recombinants identification. There exist two problems when using Serratia marcescens as a template for PCR amplification: amplified PCR products are rapidly degraded, and the results of PCR amplification are unstable. The aim of the present work was to elucidate the reasons for this. By mixing PCR products amplified from Escherichia coli DH5α with S. marcescens supernatant or pellet, we found that the DNA-degrading substance in S. marcescens is thermally resistant and present both intracellularly and extracellularly. We then determined that it is protein, and most likely S. marcescens nuclease, that degrades PCR products since the addition of SDS and EDTA can effectively inhibit or block the degradation of PCR products. By knocking out the S. marcescens nuclease encoding gene, nucA, we confirmed that the nuclease is responsible for the degradation of PCR products and the instability of PCR amplification. This work is the first to show that the S. marcescens nuclease is temporarily and partially inhibited by high temperatures during PCR and recovers rapidly at room temperature after PCR.

Mutagenic primer-based novel multiplex PCR-RFLP technique to genotype BECN1 SNPs rs10512488 and rs11552192.

Single Nucleotide Polymorphisms (SNPs) in candidate autophagy gene BECN1 could influence its functions thereby autophagy process. BECN1 noncoding SNPs were found to be significantly associated with neurodegenerative disease and type 2 diabetes mellitus. This study aimed to develop a simultaneous genotyping technique for two BECN1 SNPs (rs10512488 and rs11552192). A mutagenic primer-based approach was used to introduce a NdeI restriction site to genotype rs10512488 by Artificial-Restriction Fragment Length Polymorphism (A-RFLP) along with rs11552192 by Polymerase Chain Reaction (PCR)-RFLP. Multiplexing PCR and restriction digestion reactions were set up for simultaneous genotyping of both SNPs in 100 healthy individuals. Genotypic and allele frequencies were manually calculated, and the Hardy-Weinberg Equilibrium was assessed using the chi-square test. We successfully developed PCR and RFLP conditions for the amplification and restriction digestion of both SNPs within the same tube for genotyping. The results of genotyping by newly developed multiplexing PCR-RFLP technique were concordant with the genotypes obtained by Sanger sequencing of samples. Allelic frequencies of rs10512488 obtained were 0.15 (A) and 0.85 (G), whereas allelic frequencies of rs11552192 were 0.16 (T) and 0.84 (A). The newly developed technique is rapid, cost-effective and time-saving for large-scale applications compared to sequencing methods and would play an important role in low-income settings. For the first time, allelic frequencies of rs10512488 and rs11552192 were reported among the North Indian population.

New insights into the photophysical properties and interaction mechanisms of Janus green blue dye with polyanions and its applications in colorimetric visualization of loop-mediated isothermal amplification and polymerase chain reaction.

In this investigation, the photophysical properties and interaction mechanisms of Janus green blue (JGB) dye with polyanions were systematically studied using spectroscopic techniques. The absorption spectral analysis revealed that JGB binds cooperatively to sodium alginate, leading to dye stacking along the polymer chain. The interaction of JGB dye with DNA was characterized by the emergence of a metachromatic peak at 564 nm, indicating the formation of dye aggregates. The analysis of absorption data reveals that JGB dye interacts with DNA at multiple binding sites, including at least one high-affinity site. The AutoDock Vina based blind docking approach was used to analyze the most probable binding location of JGB dye in DNA. By making use of the DNA-induced metachromasia, a colorimetric approach was developed for the visualization of loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The LAMP-colorimetric assay, targeting the Streptococcus pneumoniae gene, demonstrated a noticeable colour change with a detection limit of 1 pg μL-1. The practical applicability was validated by detecting S. pneumoniae in artificial urine. In addition to LAMP, we tested the JGB dye based colorimetric assay for applicability in PCR reactions. The colorimetric PCR assay using the metal-responsive transcription factor (MTF-1) gene achieved a detection limit as low as 0.1 pg μL-1. The study highlights the potential of DNA binding metachromic dye to significantly enhance colorimetric assays, offering a robust and sensitive tool for molecular diagnostics.

Quantitating primer-template interactions using deconstructed PCR.

When the polymerase chain reaction (PCR) is used to amplify complex templates such as metagenomic DNA using single or degenerate primers, preferential amplification of templates (PCR bias) leads to a distorted representation of the original templates in the final amplicon pool. This bias can be influenced by mismatches between primers and templates, the locations of mismatches, and the nucleotide pairing of mismatches. Many studies have examined primer-template interactions through interrogation of the final products of PCR amplification with controlled input templates. Direct measurement of primer-template interactions, however, has not been possible, leading to uncertainty when optimizing PCR reactions and degenerate primer pools. In this study, we employed a method developed to reduce PCR bias (i.e., Deconstructed PCR, or DePCR) that also provides empirical data regarding primer-template interactions during the first two cycles of PCR amplification. We systematically examined interactions between primers and templates using synthetic DNA templates and varying primer pools, amplified using standard PCR and DePCR protocols. We observed that in simple primer-template systems, perfect match primer-template interactions are favored, particularly when mismatches are close to the 3' end of the primer. In more complex primer-template systems that better represent natural samples, mismatch amplifications can dominate, and heavily degenerate primer pools can improve representation of input templates. When employing the DePCR methodology, mismatched primer-template annealing led to amplification of source templates with significantly lower distortion relative to standard PCR. We establish here a quantitative experimental system for interrogating primer-template interactions and demonstrate the efficacy of DePCR for amplification of complex template mixtures with complex primer pools.

Streamlined instrument-free lysis for the detection of Candida auris

The continued spread of Candida auris in healthcare facilities has increased the demand for widely available screening to aid in containment and inform treatment options. Current methods of detection can be unreliable and require bulky and expensive instruments to lyse and identify fungal pathogens. Here, we present a quick, low-cost, instrument-free method for lysis of C. auris suitable for streamlined sample processing with polymerase chain reaction (PCR) detection. Chemical, thermal, and bead beating lysis techniques were evaluated for lysis performance and compatibility with nucleic acid extraction and downstream PCR reactions. Using only 10 s of manual shaking with glass beads, this method demonstrated a limit of detection (LOD) of C. auris at 500 colony forming units per mL, a 20-fold improvement compared to the LOD without manual shaking, and a 60-fold reduction in time compared to common fungal lysis kits, all while maintaining repeatability and reproducibility across multiple users. This work highlights a simple method for increasing sensitivity and reducing turnaround time of PCR-based C. auris detection and exhibits promise for integration into point-of-care platforms towards real-time triage of colonized patients.

A Multiplex PCR System of Novel Microsatellite Loci for Population Genetic Application in Walnuts.

Multiplex polymerase chain reaction (PCR) of microsatellite loci allows for simultaneous amplification of two or more pairs of primers in a single PCR reaction; hence, it is cost and time effective. However, very few attempts have been reported in non-model species. In this study, by combining a genome-based de novo development and cross-species application approach, a multiplex PCR system comprising 5 PCR reactions of 33 microsatellites consisting of 26 novel genomic and 7 literature-sourced loci was tested for polymorphisms, cross-species transferability, and the ability to assess genetic diversity and population structure of three walnut species (Juglans spp.). We found that the genome-based approach is more efficient than other methods. An allelic ladder was developed for each locus to enhance consistent genotyping among laboratories. The population genetic analysis results showed that all 33 loci were successfully transferred across the three species, showing high polymorphism and a strong genetic structure. Hence, the multiplex PCR system is highly applicable in walnut species. Furthermore, we propose an efficient pipeline to characterize and genotype polymorphic microsatellite loci. The novel toolbox developed here will aid future ecology and evolution studies in walnut and could serve as a model for other plant species.

Development and validation of a two-step assay for differentiation of Penis et testis cervi from cervi nippon Temmink and cervi elaphus Linnaeus based on specific-species polymerase chain reaction-restriction fragment length polymorphism patterns

Penis et testis cerviis one of the precious traditional Chinese medicines (TCMs) in China, which, whenauthentic, is sourcedfrom species ofCervi nippon Temmink(C. Nippon)andCervi elaphus Linnaeus(C.elaphus L).Authentication of species ofPenis et testis cerviremainstechnically challenging. This study attempted to modify the species-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP) method using restriction enzymeHpy188III for the identification ofPenis et testis cervispecies.First, thecytochrome b (Cytb)and cytochrome c oxidase subunit I (COI) genes in mitochondrial DNAfrom two authentic species and their counterfeitsor adulterantswere downloaded and analyzed with bioinformatics tools to establish the phylogenetic tree, then a series of primers were designed, and the restriction enzymesites were confirmed. Genomic DNA was extracted fromall samplesby the improved SDS method. Species-specific PCR reactions were optimizedto differentiate authentic Sika deer and ReddeerPenis et testis cervifrom their counterfeits or adulterants. PCR-RFLP using single restriction enzyme Hpy188III was performed inCytb regionsto identify the species of Sika deer and Reddeer. Simultaneously, recombination plasmid carrying thecytb fragmentcould be cloned, sequenced, and blasted.The assay specificity and sensitivity were evaluated, and30 batches of commercial samples werevalidated. Thephylogenetic treeshowed that the physico-chemical properties of Sika deer and Reddeer variants were similar.Sufficient DNA extracts were extracted from all samples used inthe improved SDS method.Establishment and optimization of a species-specific PCR reactionsand PCR-RFLPsystem were undertaken to identify deer species inPenis et testis cervisamples. Aspecific PCR-RFLP pattern with three distinct products of sizes 155, 113, and 204 bp was exhibited in Sika deerspecies,and two distinct products of 204 and 268 bp in size were evidentin Reddeerspecies,which were significantly different from the pattern of thecounterfeitsor adulterants.The distinctive PCR-RFLP fingerprint pattern using the restriction enzyme Hpy188III is a rapid and reliable assay for identification of thePenis et testis cervispecies.

Boron nitride nanoplate-based improvement of the specificity and sensitivity in loop-mediated isothermal amplification for Vibrio parahaemolyticus detection

BackgroundNucleic acid testing based on DNA amplification is gradually entering people's modern life for clinical diagnosis, food safety monitoring and infectious disease prevention. Polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) are the most powerful techniques that have been the gold standard for quantitative nucleic acid analysis. However, the high nonspecific amplification rate caused by the formation of primer dimers, hairpin structures and mismatched hybridization severely restricts their real-world applications. It is highly desirable to explore a way for improving the specificity and sensitivity of PCR and LAMP assays. ResultsIn this work, we demonstrated that a nanomaterial boron nitride nanoplate (BNNP), due to its unique surface properties, can interact with the main components of the amplification reaction, such as single stranded primers and Bst DNA polymerase, and increase the thermal conductivity of the solution. As a result, the presence of BNNPs dramatically improved the specificity of PCR and LAMP. And BNNPs maintained the specificity even after five rounds of PCR. Moreover, the sensitivity of LAMP was also enhanced by BNNPs, and the detection limit of BNNP-based LAMP was two orders of magnitude lower than that of classical LAMP. Then the BNNP-based LAMP was applied to detect Vibrio parahaemolyticus in contaminated seafood samples with high specificity and a 10-fold increase in sensitivity. SignificanceThis is the first systematic demonstration of BNNPs as a promising additive to enhance the efficiency and fidelity of PCR and LAMP amplification reactions, thereby greatly expanding the application of nucleic acid detection in a wide range of laboratory and clinical settings.

Polymorphisms and expression levels of TNP2, SYCP3, and AZFa genes in patients with azoospermia.

Azoospermia (the total absence of sperm in the ejaculate) affects approximately 10% of infertile males. Despite diagnostic advances, azoospermia remains the most challenging issue associated with infertility treatment. Our study evaluated transition nuclear protein 2 (TNP2) and synaptonemal complex protein 3 (SYCP3) polymorphisms, azoospermia factor a (AZFa) microdeletion, and gene expression levels in 100 patients with azoospermia. We investigated a TNP2 single-nucleotide polymorphism through polymerase chain reaction (PCR) restriction fragment length polymorphism analysis using a particular endonuclease. An allele-specific PCR assay for SYCP3 was performed utilizing two forward primers and a common reverse primer in two PCR reactions. Based on the European Academy of Andrology guidelines, AZFa microdeletions were evaluated by multiplex PCR. TNP2, SYCP3, and the AZFa region main gene (DEAD-box helicase 3 and Y-linked [DDX3Y]) expression levels were assessed via quantitative PCR, and receiver operating characteristic curve analysis was used to determine the diagnostic capability of these genes. The TNP2 genotyping and allelic frequency in infertile males did not differ significantly from fertile volunteers. In participants with azoospermia, the allelic frequency of the SYCP3 mutant allele (C allele) was significantly altered. Deletion of sY84 and sY86 was discovered in patients with azoospermia and oligozoospermia. Moreover, SYCP3 and DDX3Y showed decreased expression levels in the azoospermia group, and they exhibited potential as biomarkers for diagnosing azoospermia (area under the curve, 0.722 and 0.720, respectively). These results suggest that reduced SYCP3 and DDX3Y mRNA expression profiles in testicular tissue are associated with a higher likelihood of retrieving spermatozoa in individuals with azoospermia. The homozygous genotype TT of the SYCP3 polymorphism was significantly associated with azoospermia.

Validating duplex-PCR targeting ND2 for bovine and porcine detection in meat products

Food authentication is a mandatory effort to assure the fair-trade. This study developed a duplex polymerase chain reaction (PCR) from the NADH dehydrogenase subunit 2 (ND2) gene to amplify specific segments of a cattle and porcine DNA. A universal forward primer composed of nineteen base pairs (bp) (3′-CCAAACACAACTCCGAAAA-5′) and species-specific reverse primers composed of twenty (3′-CCAAACACAACTCCGAAAA-5′) and twenty-one (3′-TGGCAAGAATTAGGACGGTTA-5′) bp were used to limit the amplified DNA segment for porcine and cattle. The PCR reaction would generate a product with a profile of 168 and 227 bp, respectively. To investigate the accuracy and limit of detection, an in vitro experiment was conducted using simplex and duplex PCR on commercial meatballs randomly purchased from a commercial market in Surakarta, Indonesia. The findings of this study indicated that ND2 could be used as an alternative genetic marker for the identification of porcine and beef species in meat-derived products.