Detection Of Amplicons Research Articles

Sequence-Specific Electrochemical Genosensor for Rapid Detection of blaOXA-51-like Gene in Acinetobacter baumannii.

Acinetobacter baumannii (A. baumannii) are phenotypically indistinguishable from the Acinetobacter calcoaceticus–A. baumannii (ACB) complex members using routine laboratory methods. Early diagnosis plays an important role in controlling A. baumannii infections and this could be assisted by the development of a rapid, yet sensitive diagnostic test. In this study, we developed an enzyme-based electrochemical genosensor for asymmetric PCR (aPCR) amplicon detection of the blaOXA-51-like gene in A. baumannii. A. baumannii blaOXA-51-like gene PCR primers were designed, having the reverse primer modified at the 5′ end with FAM. A blaOXA-51-like gene sequence-specific biotin labelled capture probe was designed and immobilized using a synthetic oligomer (FAM-labelled) deposited on the working electrode of a streptavidin-modified, screen-printed carbon electrode (SPCE). The zot gene was used as an internal control with biotin and FAM labelled as forward and reverse primers, respectively. The blaOXA-51-like gene was amplified using asymmetric PCR (aPCR) to generate single-stranded amplicons that were detected using the designed SPCE. The amperometric current response was detected with a peroxidase-conjugated, anti-fluorescein antibody. The assay was tested using reference and clinical A. baumannii strains and other nosocomial bacteria. The analytical sensitivity of the assay at the genomic level and bacterial cell level was 0.5 pg/mL (1.443 µA) and 103 CFU/mL, respectively. The assay was 100% specific and sensitive for A. baumannii. Based on accelerated stability performance, the developed genosensor was stable for 1.6 years when stored at 4 °C and up to 28 days at >25 °C. The developed electrochemical genosensor is specific and sensitive and could be useful for rapid, accurate diagnosis of A. baumannii infections even in temperate regions.

Longer amplicons provide better sensitivity for electrochemical sensing of viral nucleic acid in water samples using PCB electrodes

The importance of monitoring environmental samples has gained a lot of prominence since the onset of COVID-19 pandemic, and several surveillance efforts are underway using gold standard, albeit expensive qPCR-based techniques. Electrochemical DNA biosensors could offer a potential cost-effective solution suitable for monitoring of environmental water samples in lower middle income countries. In this work, we demonstrate electrochemical detection of amplicons as long as {503},hbox {bp} obtained from Phi6 bacteriophage (a popular surrogate for SARS-CoV-2) isolated from spiked lake water samples, using ENIG finish PCB electrodes with no surface modification. The electrochemical sensor response is thoroughly characterised for two DNA fragments of different lengths ({117},hbox {bp} and {503},hbox {bp}), and the impact of salt in PCR master mix on methylene blue (MB)-DNA interactions is studied. Our findings establish that length of the DNA fragment significantly determines electrochemical sensitivity, and the ability to detect long amplicons without gel purification of PCR products demonstrated in this work bodes well for realisation of fully-automated solutions for in situ measurement of viral load in water samples.

Investigation and validation of labelling loop mediated isothermal amplification (LAMP) products with different nucleotide modifications for various downstream analysis

Loop mediated isothermal amplification (LAMP) is one of the best known and most popular isothermal amplification methods. It's simplicity and speed make the method particularly suitable for point-of-care diagnostics. Nevertheless, false positive results remain a major drawback. Many (downstream) applications are known for the detection of LAMP amplicons like colorimetric assays, in-situ LAMP or CRISPR-Cas systems. Often, modifications of the LAMP products are necessary for different detection applications such as lateral flow assays. This is usually achieved with pre-modified primer. The aim of this study is to evaluate amplicon labelling with different modified nucleotides such as Cy5-dUTP, biotin-dUTP and aminoallyl-dUTP as an alternative to pre-labelled primers. To realise this, the effects on amplification and labelling efficiency were studied as a function of molecule size and nucleotide amount as well as target concentration. This research shows that diverse labelling of LAMP amplicons can be achieved using different, modified nucleotides during LAMP and that these samples can be analysed by a wide range of downstream applications such as fluorescence spectroscopy, gel electrophoresis, microarrays and lateral flow systems. Furthermore, microarray-based detection and the ability to identify and distinguish false positives were demonstrated as proof of concept.

Hapten-labeled fusion-polymerase chain reaction of multiple marker genes for the application of immunochromatographic test

A variety of methods have been reported using polymerase chain reaction (PCR)-based nucleic acid testing (NAT) because of its potential to be used in highly sensitive inspection systems. Among these NATs, fusion-PCR (also called as overlap-extension-PCR) has been focused on this study and adopted to generate the fused amplicon composed of plural marker gene fragments for detection. Generally, conventional agarose gel electrophoresis followed by gel staining is employed to check the PCR results. However, these are time-consuming processes that use specific equipment. To overcome these disadvantages, the immunochromatographic test (ICT) for the detection of PCR amplicons with hapten-labels that were generated by PCR using hapten-labeled primers was also adopted in this study. Based on these concepts, we constructed the systems of hapten-labeled fusion-PCR (HL-FuPCR) followed by ICT (HL-FuPCR-ICT) for the two and three marker genes derived from pathogenic microbe. As a result, we successfully developed a two marker genes system for the pathogenic influenza A virus and a three marker genes system for the penicillin-resistant Streptococcus pneumoniae. These detection systems of HL-FuPCR-ICT are characterized by simple handling and rapid detection within few minutes, and also showed the results as clear lines. Thus, the HL-FuPCR-ICT system introduced in this study has potential for use as a user-friendly inspection tool with the advantages especially in the detection of specific strains or groups expressing the characteristic phenotype(s) such as antibiotic resistance and/or high pathogenicity even in the same species.

Detection of porcine DNA in food using direct asymmetric PCR and catalyzed hairpin assembly fluorescent biosensor: A novel assay for halal food analysis

Adulteration of halal-certified food with pork are often reported. Using direct PCR results in cost- and time-saving benefits, and allows flexibility in amplicon detection. Here, we aimed to develop and validate a novel, direct asymmetric PCR and catalyzed hairpin assembly fluorescent biosensor (daPCR-CHA-FB) with a portable LED lightbox for naked eye detection of porcine DNA in raw and processed food. We tested the assay's reproducibility, specificity, sensitivity, real-world applicability, and single-blind performance. Using only 1 mm3 samples, reproducibility test with 38 raw pork samples showed 2.6% false negative rate and 0.0% false positive rate. The lowest detection limits were 0.01 ng for porcine DNA and 0.1% for binary pork-and-beef mixture. No cross-reaction with 16 other species was observed. One-hundred market samples showed no false negative for pork-listed samples, while two samples declared as non-pork showed a positive reaction. The assay was simple, sensitive, specific, safe, and robust. It could be used by law enforcement agencies and small-scale laboratories to investigate contamination and adulteration of halal foods.

Plasmonic LAMP: Improving the Detection Specificity and Sensitivity for SARS-CoV-2 by Plasmonic Sensing of Isothermally Amplified Nucleic Acids.

The ability to detect pathogens specifically and sensitively is critical to combat infectious diseases outbreaks and pandemics. Colorimetric assays involving loop‐mediated isothermal amplification (LAMP) provide simple readouts yet suffer from the intrinsic non‐template amplification. Herein, a highly specific and sensitive assay relying on plasmonic sensing of LAMP amplicons via DNA hybridization, termed as plasmonic LAMP, is developed for the severe acute respiratory syndrome‐related coronavirus 2 (SARS‐CoV‐2) RNA detection. This work has two important advances. First, gold and silver (Au–Ag) alloy nanoshells are developed as plasmonic sensors that have 4‐times stronger extinction in the visible wavelengths and give a 20‐times lower detection limit for oligonucleotides over Au counterparts. Second, the integrated method allows cutting the complex LAMP amplicons into short repeats that are amendable for hybridization with oligonucleotide‐functionalized Au–Ag nanoshells. In the SARS‐CoV‐2 RNA detection, plasmonic LAMP takes ≈75 min assay time, achieves a detection limit of 10 copies per reaction, and eliminates the contamination from non‐template amplification. It also shows better detection specificity and sensitivity over commercially available LAMP kits due to the additional sequence identification. This work opens a new route for LAMP amplicon detection and provides a method for virus testing at its early representation.

Acoustic Array Biochip Combined with Allele-Specific PCR for Multiple Cancer Mutation Analysis in Tissue and Liquid Biopsy.

Regular screening of point mutations is of importance to cancer management and treatment selection. Although techniques like next-generation sequencing and digital polymerase chain reaction (PCR) are available, these are lacking in speed, simplicity, and cost-effectiveness. The development of alternative methods that can detect the extremely low concentrations of the target mutation in a fast and cost-effective way presents an analytical and technological challenge. Here, an approach is presented where for the first time an allele-specific PCR (AS-PCR) is combined with a newly developed high fundamental frequency quartz crystal microbalance array as biosensor for the amplification and detection, respectively, of cancer point mutations. Increased sensitivity, compared to fluorescence detection of the AS-PCR amplicons, is achieved through energy dissipation measurement of acoustically "lossy" liposomes binding to surface-anchored dsDNA targets. The method, applied to the screening of BRAF V600E and KRAS G12D mutations in spiked-in samples, was shown to be able to detect 1 mutant copy of genomic DNA in an excess of 104 wild-type molecules, that is, with a mutant allele frequency (MAF) of 0.01%. Moreover, validation of tissue and plasma samples obtained from melanoma, colorectal, and lung cancer patients showed excellent agreement with Sanger sequencing and ddPCR; remarkably, the efficiency of this AS-PCR/acoustic methodology to detect mutations in real samples was demonstrated to be below 1% MAF. The combined high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness, and compatibility with routine workflow, make this approach a promising tool for implementation in clinical oncology labs for tissue and liquid biopsy.

Screening for Group A Streptococcal Disease via Solid-State Nanopore Detection of PCR Amplicons.

Single-molecule detection methods are becoming increasingly important for diagnostic applications. Practical early detection of disease requires sensitivity down to the level of single copies of the targeted biomarkers. Of the candidate technologies that can address this need, solid-state nanopores show great promise as digital sensors for single-molecule detection. Here, we present work detailing the use of solid-state nanopores as downstream sensors for a polymerase chain reaction (PCR)-based assay targeting group A streptococcus (strep A), which can be readily extended to detect any pathogen that can be identified with a short nucleic acid sequence. We demonstrate that with some simple modifications to the standard PCR reaction mixture, nanopores can be used to reliably identify strep A in clinical samples. We also discuss methodological best practices for both adapting PCR-based assays to solid-state nanopore readout and analytical approaches by which to decide on sample status.

Genetic diagnosis of ADPKD

Sequencing techniques represent the gold standard in molecular diagnosis ofautosomal dominant polycystic kidney disease (ADPKD), being in this case able to accurately and concretely highlight mutations at the genome level. The PKD1 gene mutation detection technique using the Real Time PCR technique and LNA hydrolysis probes exploits the specific behavior of hybridization LNA probes under the conditions of maximum complementarity with the target sequence and consequently the theoretical possibility of designing two such probes, one for the wild genotype (WT) and the other to identify the point mutation (MUT). The technique of identifying the PKD1 gene mutation using the Real Time PCR technique and FRET type hydrolysis probes consists of a derivation of the Real Time PCR technique, with the detection of amplicons by means of fluorescence with hybridization probes. The technique of identifying the PKD1 gene mutation using the Real Time PCR technique coupled with HRM (High Resolution Melting) involves one of the variants of the Real Time PCR technique, namely the interpretation of the results according to the melting temperature.

DNA nanomachine for visual detection of structured RNA and double stranded DNA.

Visual detection of ssRNA and dsDNA amplicons was achieved at room temperature without the need for a probe-analyte annealing stage. This approach uses a DNA nanostructure equipped with two analyte-binding arms. Highly selective binding of the third arm leads to the formation of a G-quadruplex structure capable of changing the solution color.

Recent advances in loop-mediated isothermal amplification (LAMP) for rapid and efficient detection of pathogens.

Loop-mediated isothermal amplification (LAMP) method has been demonstrated to bea reliable and robust method for detection and identification of viral and microbial pathogens. LAMP method of amplification, coupled with techniques for easy detection of amplicons, makes a simple-to-operate and easy-to-read molecular diagnostic tool for both laboratory and on-field settings. Several LAMP-based diagnostic kits and assays have been developed that are specifically targeted against a variety of pathogens. With the growing needs of the demanding molecular diagnostic industry, many technical advances have been made over the years by combining the basic LAMP principle with several other molecular approaches like real-time detection, multiplex methods, chip-based assays.This has resulted in enhancing thethe sensitivity and accuracy of LAMP for more rigorous and wide-ranging pathogen detection applications. This review summarizes the current developments in LAMP technique and their applicability in present and future disease diagnosis.

Dual-color blending based visual LAMP for food allergen detection: A strategy with enlarged color variation range and contrast

Food allergy has been a serious public health problem around the world. Its prevention relies heavily on the effective avoidance of any contaminated food, making clear and accurate detection very important. LAMP is one of the most potent methods for allergen rapid detection. However, its current colorimetric readouts usually have low color contrast and narrow color variation range. Thus, here we proposed a strategy based on color evolution to enlarge the variation range as well as the contrast to improve its suitability for naked-eye observation. By simply blending two commonly used color change processes during amplification, a wider color variation window, and a near contrast color change, purple-to-green with a hues difference of 10 were obtained. Three important allergens (walnuts, hazelnuts, and peanuts) were tested with a comparable sensitivity towards fluorescent real-time LAMP. Its feasibility for practical use has also been studied. This simple but effective strategy provides a new idea for the colorimetric detection of LAMP amplicons and can be applied to various fields.

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens.

Rapid, sensitive, and timely diagnostics are essential for protecting plants from pathogens. Commonly, PCR techniques are used in laboratories for highly sensitive detection of DNA/RNA from viral, viroid, bacterial, and fungal pathogens of plants. However, using PCR-based methods for in-field diagnostics is a challenge and sometimes nearly impossible. With the advent of isothermal amplification methods, which provide amplification of nucleic acids at a certain temperature and do not require thermocyclic equipment, going beyond the laboratory has become a reality for molecular diagnostics. The amplification stage ceases to be limited by time and instruments. Challenges to solve involve finding suitable approaches for rapid and user-friendly plant preparation and detection of amplicons after amplification. Here, we summarize approaches for in-field diagnostics of phytopathogens based on different types of isothermal amplification and discuss their advantages and disadvantages. In this review, we consider a combination of isothermal amplification methods with extraction and detection methods compatible with in-field phytodiagnostics. Molecular diagnostics in out-of-lab conditions are of particular importance for protecting against viral, bacterial, and fungal phytopathogens in order to quickly prevent and control the spread of disease. We believe that the development of rapid, sensitive, and equipment-free nucleic acid detection methods is the future of phytodiagnostics, and its benefits are already visible.

Microfluidic Biosensor for Rapid Nucleic Acid Quantitation Based on Hyperspectral Interferometric Amplicon-Complex Analysis.

Nucleic acid detection plays a vital role in both biomedical research and clinical medicine. The temperature circulation changes of the widely used polymerase chain reaction technique are time-consuming and technically challenging for system development. Recombinase polymerase amplification (RPA) is an isothermal method for rapid nucleic acid detection. However, current RPA amplicon detection methods are complicated and expensive and easily generate false positives, restricting the promotion of RPA techniques. In this work, a hyperspectral interferometric amplicon-complex quantitation method is presented, combined with asymmetric dipole complex strategy optical scattering analysis. GelRed dye was utilized to form amplicon-complex particles, and the Fourier domain spectrum computation contributed to complex scattering quantitation. With this method, a supporting microfluidic chip and automatic system were developed to achieve integrated, rapid, quantitative, and miniscule nucleic acid detection. The Plasmodium falciparumdhfr gene was utilized as an example for targeted nucleic acid quantitation and single nucleotide polymorphism detection. The total reaction time was decreased to merely 20 min, and the limit of detection was only 3.17 ng/μL. The minimum measurable concentration of target was 1.68 copies/μL, 31.67 times more sensitive than turbidity detection, and the single reaction chamber was only 9.33 μL. No scattering increase occurred for template-free control, and thus, false positives caused by primer dimers and nonspecific products could be avoided. The experimental results prove that the provided method and system can detect single-base mutations in the dhfr gene and is a reasonable technique for rapid, automatic, and low-cost nucleic acid detection.

Rapid identification of Atlantic salmon (Salmo salar) based on loop-mediated isothermal amplification (LAMP) using self-quenching fluorogenic approach

Loop-mediated isothermal amplification (LAMP) has emerged as an important diagnostic tool for fish authenticity purpose. While the sequence-independent detection methods greatly increased the likelihood of detecting false-positive signals. Instead, target-specific detection of LAMP amplicons can be achieved through the use of target-specific probes or modified primers as biorecognition elements. The present work selected Salmo salar as a case study, and developed a novel LAMP assay coupled with the self-quenching approach for accurate species identification.Specifically, the loop primer (LB-6) was identified as the candidate primer to design the self-quenching element. The fluorophore attached to LB-6 is self-quenched in unbound state. After binding to the dumb bell shaped DNA specifically, the FAM fluorophore is de-quenched, resulting in the fluorescence release. With the novel assay, as little as 5 pg of S. salar DNA could be detected. Moreover, the self-quenching assay embraces a high tolerance to impurities and the positive LAMP results can always be obtained, regardless of the DNA extraction methods and sample treatments. While significant difference with the Ct value according to both the extraction methods and sample treatments, highlighted also an improved LAMP amplification using DNA with higher purity.

Loop-Mediated Isothermal Amplification Detection of SARS-CoV-2 and Myriad Other Applications.

As the second year of the COVID-19 pandemic begins, it remains clear that a massive increase in the ability to test for SARS-CoV-2 infections in a myriad of settings is critical to controlling the pandemic and to preparing for future outbreaks. The current gold standard for molecular diagnostics is the polymerase chain reaction (PCR), but the extraordinary and unmet demand for testing in a variety of environments means that both complementary and supplementary testing solutions are still needed. This review highlights the role that loop-mediated isothermal amplification (LAMP) has had in filling this global testing need, providing a faster and easier means of testing, and what it can do for future applications, pathogens, and the preparation for future outbreaks. This review describes the current state of the art for research of LAMP-based SARS-CoV-2 testing, as well as its implications for other pathogens and testing. The authors represent the global LAMP (gLAMP) Consortium, an international research collective, which has regularly met to share their experiences on LAMP deployment and best practices; sections are devoted to all aspects of LAMP testing, including preanalytic sample processing, target amplification, and amplicon detection, then the hardware and software required for deployment are discussed, and finally, a summary of the current regulatory landscape is provided. Included as well are a series of first-person accounts of LAMP method development and deployment. The final discussion section provides the reader with a distillation of the most validated testing methods and their paths to implementation. This review also aims to provide practical information and insight for a range of audiences: for a research audience, to help accelerate research through sharing of best practices; for an implementation audience, to help get testing up and running quickly; and for a public health, clinical, and policy audience, to help convey the breadth of the effect that LAMP methods have to offer.

A cloth-based hybridization array system for rapid detection of the food- and waterborne protozoan parasites Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii

Protozoan parasites in food or water samples are generally detected using microscopy or PCR followed by Sanger sequencing. However, microscopy is subjective, requires a high degree of expertise and has limited sensitivity, while DNA sequencing requires expensive and specialized equipment and facilities. This study describes a cloth-based hybridization array system (CHAS) that is an alternative to Sanger sequencing to confirm PCR-positive samples. CHAS is an inexpensive, rapid and reliable method for the simultaneous detection of multiple protozoan parasite species based on the colorimetric detection of PCR amplicons on a polyester cloth. PCR primers and CHAS hybridization probes were developed to detect the protozoan parasites Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii. In addition, CHAS probes were designed for the differentiation of G. duodenalis Assemblages A and B. In artificially contaminated fresh produce (lettuce, parsley) and water samples (river water, wastewater), this CHAS assay allowed for the successful detection of G. duodenalis, Cryptosporidium spp., and T. gondii. The present study demonstrates that the CHAS detection method is a simple and inexpensive alternative to DNA sequencing for the confirmation of PCR-positive results in laboratories testing for parasites in food or water samples. This assay may also be beneficial in developing countries, where DNA sequencing facilities may not be readily available.

Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons

BackgroundRapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection.ResultsHere we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in “single pot” reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes.ConclusionsLAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.

Closed-tube field-deployable loop-mediated isothermal amplification (LAMP) assay based on spore wall protein (SWP) for the visual detection of Enterocytozoon hepatopenaei (EHP)

Hepatopancreatic microsporidiosis (HPM) is an infectious shrimp disease caused by the microsporidian Enterocytozoon hepatopenaei (EHP). In recent years, the widespread occurrence of EHP poses a significant challenge to the shrimp aquaculture industry. Early, rapid and accurate diagnosis of EHP infection is very much essential for the control of HPM crop-related losses. Loop-mediated isothermal amplification (LAMP) is a robust, sensitive, cost-effective disease diagnostic technique. Here, we demonstrate an improved, simple, closed-tube, colorimetric EHP LAMP diagnostic assay. LAMP assay was illustrated with the specific EHP spore wall protein (SWP) gene primers. Naked eye visual detection of LAMP amplicons was achieved using Hydroxy naphthol blue (HNB) or Phenol red dye without opening the tubes. This LAMP assay is efficient in detecting the EHP pathogen in all clinical samples include shrimp hepatopancreas, FTA card samples, feces, pond water, and soil. Also, the elution of EHP DNA from FTA cards was demonstrated within 17 min using a simple dry bath. In clinical evaluation, the visual LAMP assay established 100% diagnostic sensitivity and 100% diagnostic specificity. The visual LAMP assay is rapid, can detect the EHP pathogen within 40 min using a simple dry bath, and does not require any expensive instruments and technical proficiency. In conclusion, this visual LAMP protocol is a user-friendly, specific assay that can be conceivably operated at the farm-site/ resource-limited settings by the farmer himself with simple equipment.

Генотипування лістерій за допомогою полімеразної ланцюгової реакції (ПЛР) та його епідеміологічне значення

The purpose of the study. The article covers the issues of genotyping of listeria by polymerase chain reaction (PCR) and its epidemiological significance. It is known that molecular genetic methods allow to detect specific microbial pathogens, virulence markers, antimicrobial resistance genes faster and with greater sensitivity than traditional culture methods. Therefore, the development of detection methods and genotyping by polymerase chain reaction (PCR) is relevant. Materials and methods. For the detection and genotyping of Listeria, the technology of DNA chips is becoming increasingly important, which can significantly expand the possibilities of molecular detection. Chip technology can be used to simultaneously identify a whole range of pathogenic microorganisms, to determine genetic virulence markers, the relationship to antibiotics, subtyping, as well as to determine the quality of microorganisms in samples. A simplified version of DNA chip technology is multiplex (numerical) PCR, which is used to detect and genotype listeria. Studies have shown that to detect Listeria spp. using a polymerase chain reaction, it is advisable to use the gene iap (invasive associated protein), known for 6 species of listeria, which encodes a protein P 60 that is common to all species of listeria, including L.murrayi. Computer analysis revealed areas with 100% homology, from which primers were selected for PCR detection of all types of listeria. Areas of genomes characterized by 100% homology were selected for further analysis and labeling of primer sets. The sequences of the constructed primers List 1 and List 2 allowed to identify 6 species of Listeria (L. monocytogenes, L. innocua, L. ivanovii, L. grayi, L. seeligeri, L. welshimeri). Increasing the length of the primer leads to the increasing of specificity of PCR analysis. The greater the length of the primer, the smaller the specific gravity of one error of the unpaired nucleotide. The degree of primer homology is a key parameter that indicates the "quality" of a set of primers. Results and discussion. It is established that a significant disadvantage of the vast majority diagnosed using PCR test systems is the lack of internal control of amplification. The negative result of PCR analysis may be due to the absence in the clinical material of a fragment of the Listeria genome, and the fact that the PCR product was not synthesized for other reasons. They may be as the following ones: operator errors, erroneously determined reaction mixture concentrations and PCR temperature parameters. False-negative results can also be caused by factors that inhibit thermostable DNA polymerase. In its turn, such inhibition of the enzyme responsible for amplification is caused by a very large amount of DNA - template, pre-treatment of clinical samples. It has been shown that 80% of clinical specimens contain a substance that inhibits DNA polymerase. Therefore, it is necessary to use internal control, the positive result of the reaction of which indicates the successful amplification, that is the absence of false positive results. Conclusion. There are several reasons why the accuracy of PCR analysis does not reach 100%. Accuracy depends on the technology (variety) of PCR - the method used (ordinary or fluorescent), detection of amplicons, PCR homogeneous or nested, nested in one test tube or in two test tubes, as well as the level of quality of the survey (primarily on the technical parameters of the amplifier). The test systems used can be used for PCR detection and are recommended as standard primer sets for the detection and cross-species testing of listeria, which is important for the timely implementation of appropriate anti-epidemic measures in listeriosis