Multiplex PCR System Research Articles

Culture-Dependent and -Independent Wastewater Surveillance for Multiple Pathogenic Yeasts

Wastewater surveillance is a promising tool to monitor potential outbreaks and determine the disease burden within a community. This system has been extensively used to monitor polio and COVID-19 infection levels, yet few attempts have been made to apply it to monitoring pathogenic yeast. This study aimed to investigate the application of wastewater surveillance for potentially pathogenic yeast in wastewater treatment plant influent. This was done by comparing culture-dependent data with culture-independent data and investigating the fluconazole concentration in wastewater. Additional studies on the growth of isolated strains were conducted. We found that a multiplex PCR system to detect multiple yeasts holds promise as a molecular detection tool for wastewater surveillance. Culture-dependent results indicated that Candida spp. specifically C. krusei and C. glabrata, were most prominent. Growth studies supported that these species grow well in this environment while the less frequently isolated yeasts grew poorly. The data from culture-dependent and independent techniques showed some correlation, with similar species being identified with both, further promoting the use of molecular tools for surveillance. This study highlights the presence of potentially pathogenic yeasts in wastewater, which may indicate the prevalence of these yeasts in the environment or community. This wastewater may also be a potential source of infection for persons encountering it due to poor wastewater management.

Evaluation of the DiaSorin NxTAG gastrointestinal pathogen panel (GPP) and its integration into routine diagnostics.

This study evaluated the performance of the DiaSorin NxTAG GPP multiplex PCR system, the advanced successor of the Luminex-xTAG-GPP, in comparison to classical diagnostic techniques for stool samples. In a testing phase, we investigated 505 specimens using both the DiaSorin system and our conventional approach consisting of culture techniques, PCR detection and microscopic analysis. The multiplex PCR system proved to be superior in sensitivity, time efficiency, and pathogen range. The analysis of 2,462 stool samples after the system's implementation into routine diagnostics further confirmed its effectiveness. However, we encountered several Salmonella-positive samples, which could neither be confirmed by culture techniques nor by an independently performed Salmonella-specific PCR. This let us to permanently incorporate a confirmatory PCR assay for Salmonella-positive samples, in order to avoid false positives. Our findings indicate that the DiaSorin NxTAG GPP is a reliable and efficient diagnostic tool for most gastrointestinal pathogens, with the exception of Salmonella.

A Multiplex PCR System for the Detection and Quantification of Four Genetically Modified Soybean Events

A Multiplex PCR System for the Detection and Quantification of Four Genetically Modified Soybean Events

Diagnostic advances in sepsis: A comparative analysis of syndromic approaches and molecular biology techniques in clinical bacteremia

Sepsis remains a leading cause of morbidity and mortality. This retrospective study, conducted at Avicenne Military Hospital in Marrakech over ten years, analyzed 3721 blood cultures, identifying 420 cases of bacteremia (11.3% positivity rate). The predominant isolates included Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Klebsiella spp., and Acinetobacter baumannii, with significant resistance patterns observed. Notably, 50% of S. aureus were methicillin-resistant (MRSA), while extended-spectrum beta-lactamase (ESBL) production was found in 30% of E. coli and Klebsiella pneumoniae isolates. The FilmArray multiplex PCR system was utilized to enhance rapid pathogen identification, reducing turnaround time from 72 hours to under 2 hours, and demonstrated high concordance with conventional cultures. The molecular diagnostics identified various resistant strains, including carbapenemase-producing organisms in K. pneumoniae and multidrug-resistant A. baumannii, underscoring the critical need for effective antimicrobial stewardship. Overall, this study emphasizes the evolving epidemiology of bacteremia, the rising antimicrobial resistance, and the utility of rapid molecular diagnostics in guiding timely and appropriate treatment, ultimately aiming to improve patient outcomes in sepsis management.

Developing a Microsatellite Polymerase Chain Reaction System for Small Yellow Croaker (Larimichthys polyactis) and Its Application in Parentage Assignment.

(1) Background: The small yellow croaker, an economically important fish in East Asia, has been subjected to population declines due to overfishing and environmental pressures. The development of effective breeding programs is considered crucial for the species, and accurate parentage assignment is deemed essential for such programs. (2) Methods: The assembled reference genome of the small yellow croaker was utilized to select highly polymorphic microsatellite markers. A multiplex PCR system was optimized for the simultaneous amplification of these markers. The system's accuracy was validated using controlled mating pairs and subsequently applied to a group mating scenario. (3) Results: The developed multiplex PCR system demonstrated high accuracy in assigning offspring to their parents in both the controlled and group mating scenarios. (4) Conclusions: The system is presented as a valuable tool for pedigree management, selective breeding, and conservation efforts for the small yellow croaker, facilitating sustainable aquaculture practices and genetic improvement.

Development of a Multiplex RT-PCR for Simultaneous Detection of Five Actinidia Viruses

Kiwifruit (Actinidia spp.) is a perennial fruit tree, and the fruit of kiwifruit is economically and nutritionally important worldwide. To date, approximately 23 species of kiwifruit viruses have been reported worldwide. As for the detection method for kiwifruit viruses, previous reports mostly used the single RT-PCR detection method. In the detection of kiwifruit viruses, multiplex RT-PCR has the advantages of being fast, reliable and inexpensive. In this study, a stable, efficient and reliable multiplex RT-PCR method for the detection of the five most common kiwifruit viruses was established. The concentrations of Mg2+ and HS-Taq and the annealing temperature in the multiplex PCR system were optimized. The results indicate that the optimal annealing temperature was 56 °C; the optimal concentration of added Mg2+ was 2 mM; and the optimal concentration of HS-Taq was 1.0 U/μL. The stability of the optimized multiplex RT-PCR system was verified by field sample testing, and the results showed that the multiplex RT-PCR system was stable, efficient and reliable. This will provide much convenience for the detection of kiwifruit viruses in the future.

Genetic diversity and DNA fingerprinting of rice varieties of Manipur using microsatellite markers

Rice variety differentiation was based on morphological descriptors (DUS) before the advent of genomic and proteomic technology. DNA fingerprinting is becoming an important molecular marker approach because of its wide application in varietal protection, classification, and conflict settlement. In the present study, genetic diversity and variability among the 11 rice varieties of Manipur at the molecular level was studied using 42 SSR markers. The genotypic data of the SSR markers revealed four sub-populations. Cluster analysis indicated the close similarity between RC Maniphou 12 and RC Maniphou 15. To create a DNA fingerprinting database for 11 rice varieties, three separate multiplex PCR systems using different combinations of 11 polymorphic SSR markers based on amplicon size were successfully developed . These were able to distinguish the distinctness of all the eleven released rice varieties.

The development of novel genome-SSRs, multiplex PCR panels, and allelic ladders for parentage identification in Tachypleus tridentatus

Tachypleus tridentatus, one of the oldest animal species on Earth, with significant biological and ecological value, its population sizes have declined due to overfishing. Stock enhancement technology has been used to protect T. tridentatus, however, its efficiency needs to be addressed. In the present study, a parentage identification system was developed. We identified 235,336 SSRs and 182,394 primer pairs were designed. Out of 321 synthesized primers, 14 highly polymorphic primers were selected to reveal the genetic diversity of 110 T. tridentatus. SSRs were amplified with the 14 developed primers, which were fluorescently labeled, and the resulting PCR products were analyzed using the ABI DNA Analyzer 3730xl. The number of alleles per locus, the polymorphism information content, the observed heterozygosity, and the expected heterozygosity ranged from 7 to 48, 0.581 to 0.963, 0.636 to 0.909, and 0.645 to 0.969, respectively. The cumulative exclusion probability with both unknown parents, with only unknown parents, or with both known parents was >99.9999% for the 14 loci. Furthermore, to facilitate data exchange across laboratories and platforms, multiplex PCR detection, SSR fragment size ladders, and parentage identification were employed. Out of the 14 primers, 6 optimal primers were selected for multiplex PCR validation, and two sets of triplex systems were established. Three thousand clones for 160 alleles of the six primers were selected for plasmid extraction and sequencing, and 600 correct plasmids were obtained for new T. tridentatus SSR fragment size ladder construction. Two pairs of parents with 12 offspring each were used for parentage identification by the multiplex PCR systems and ladders, the cumulative average exclusion probability of these 6 primers reached 99.9999% among parents and their offsprings, and was 0% in nonparent-offspring group. The parentage identification system constructed in this study can serve as a tool for monitoring the survival rates of T. tridentatus in stock enhancement programs.

Simultaneous detection of eight dairy-derived components using multiplex PCR combined with gene membrane chip

Food adulteration poses a significant risk to food safety, especially in the case of milk and dairy products, which have garnered substantial attention from regulatory authorities. In this study, we developed a method for simultaneous detection of eight animal-derived components in milk and dairy products, employing multiplex PCR combined with gene membrane chip. These components encompass cow, yak, buffalo, goat, sheep, horse, donkey, and camel. Eight dairy-derived animal-specific probes, along with eukaryotic internal reference and plant-derived internal reference probes, were designed and immobilized on nylon membranes for hybridization with PCR products, enabling source identification. Utilizing reverse dot blot hybridization, the gene membrane chip facilitates the determination of eight dairy-derived components and the presence of plant-derived components. The method demonstrated an absolute sensitivity of 0.01 ng in the multiplex PCR and membrane chip hybridization system, with a detection limit of 0.1%, meeting market supervision requirements. Upon analysis of 75 commercially available dairy products, a mislabeling rate of 29.33% was revealed. Comparative analysis with conventional PCR and real-time quantitative PCR demonstrated the method's high specificity, good repeatability, and significantly improved detection throughput, thereby enhancing the screening efficiency for adulterants in dairy products and the targeted detection capability.

Development of a Multiplex PCR Assay for the Detection of Tomato Wilt Caused by Coinfection of Fusarium brachygibbosum, Fusarium oxysporum, and Ralstonia solanacearum Based on Comparative Genomics.

Tomato is consumed worldwide as fresh or processed food products. However, soilborne diseases of tomato plants caused by coinfection of various pathogens result in great economic losses to the tomato industry. It is difficult to accurately identify and diagnose soilborne diseases of tomato plants caused by pathogen complexes. In this study, we investigated field diseases of tomato plants by pathogen isolation and molecular identification and found that tomato wilt was caused by coinfection of Fusarium brachygibbosum, F. oxysporum, and Ralstonia solanacearum. Therefore, developing a method for simultaneous detection of DNA from F. brachygibbosum, F. oxysporum, and R. solanacearum is of great importance to efficiently and accurately monitor disease development at different growth stages of tomato plants. In this study, we performed a comparative genomic analysis of F. brachygibbosum, F. oxysporum, and R. solanacearum and determined the primer sets for simultaneous detection of DNA from these target pathogens. Then, we tested the reagent and condition parameters of multiplex PCR, including primers, dNTP and Mg2+ concentrations, and annealing temperatures, to determine the optimal parameters of a multiplex PCR system. We evaluated the specificity, sensitivity, and stability of the multiplex PCR system based on the optimized reaction conditions. The multiplex PCR system can specifically identify 13 target pathogens from 57 different fungal and bacterial pathogens, at the lower detection limit of the three target pathogens at concentrations of 100 pg/μl. In addition, we can accurately identify the three pathogens in tomato plants using the optimized multiplex PCR method. These results demonstrated that the multiplex PCR method developed in this study can simultaneously detect DNA from F. brachygibbosum, F. oxysporum, and R. solanacearum in a single PCR system to accurately identify and diagnose the pathogen causing tomato wilt.

A multiplex direct PCR method for the rapid and accurate discrimination of three species of spider mites (Acari: Tetranychidae) in fruit orchards in Beijing.

Spider mites (Acari: Tetranychidae) are polyphagous pests of economic importance in agriculture, among which the two-spotted spider mite Tetranychus urticae Koch has spread widely worldwide as an invasive species, posing a serious threat to fruit tree production in China, including Beijing. The hawthorn spider mite, Amphitetranychus viennensis Zacher, is also a worldwide pest of fruit trees and woody ornamental plants. The cassava mite, Tetranychus truncatus Ehara, is mainly found in Asian countries, including China, Korea and Japan, and mainly affects fruit trees and agricultural crops. These three species of spider mites are widespread and serious fruit tree pests in Beijing. Rapid and accurate identification of spider mites is essential for effective pest and plant quarantine in Beijing orchard fields. The identification of spider mite species is difficult due to their limited morphological characteristics. Although the identification of insect and mite species based on PCR and real-time polymerase chain reaction TaqMan is becoming increasingly common, DNA extraction is difficult, expensive and time-consuming due to the minute size of spider mites. Therefore, the objective of this study was to establish a direct multiplex PCR method for the simultaneous identification of three common species of spider mites in orchards, A. viennensis, T. truncatus and T. urticae, to provide technical support for the differentiation of spider mite species and phytosanitary measures in orchards in Beijing. Based on the mitochondrial cytochrome c oxidase subunit I (COI) of the two-spotted spider mite and the cassava mite and the 18S gene sequence of the hawthorn spider mite as the amplification target, three pairs of specific primers were designed, and the primer concentrations were optimized to establish a direct multiplex PCR system for the rapid and accurate discrimination of the three spider mites without the need for DNA extraction and purification. The method showed a high sensitivity of 0.047ng for T. truncatus and T. urticae DNA and 0.0002ng for A. viennensis. This method eliminates the DNA extraction and sequencing procedures of spider mite samples, offers a possibility for rapid monitoring of multiple spider mites in an integrated microarray laboratory system, reducing the time and cost of leaf mite identification and quarantine monitoring in the field.

Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B Virus.

A sensitive and specific multiplex fluorescence rapid detection method was established for simultaneous detection of SARS-CoV-2, influenza A virus, and influenza B virus in a self-made device within 30 min, with a minimum detection limit of 200 copies/mL. Based on the genome sequences of SARS-CoV-2, influenza A virus (FluA), and influenza B virus (FluB) with reference to the Chinese Center for Disease Control and Prevention and related literature, specific primers were designed, and a multiplex fluorescent PCR system was established. The simultaneous and rapid detection of SARS-CoV-2, FluA, and FluB was achieved by optimizing the concentrations of Taq DNA polymerase as well as primers, probes, and Mg2+. The minimum detection limits of the nucleic acid rapid detection system for SARS-CoV-2, FluA, and FluB were evaluated. By optimizing the amplification system, the N enzyme with the best amplification performance was selected, and the optimal concentration of Mg2+ in the multiamplification system was 3 mmol/L; the final concentrations of SARS-CoV-2 NP probe and primer were 0.15 μmol/L and 0.2 μmol/L, respectively; the final concentrations of SARS-CoV-2 ORF probe and primer were both 0.15 μmol/L; the final concentrations of FluA probe and primer were 0.2 μmol/L and 0.3 μmol/L, respectively; the final concentrations of FluB probe and primer were 0.15 μmol/L and 0.25 μmol/L, respectively. A multiplex real-time quantitative fluorescence RT-PCR system for three respiratory viruses of SARS-CoV-2, FluA, and FluB was established with a high amplification efficiency and sensitivity reaching 200 copies/mL for all samples. Combined with the automated microfluidic nucleic acid detection system, the system can achieve rapid detection in 30 minutes.

Development of Pollen Parent Cultivar-Specific SCAR Markers and a Multiplex SCAR-PCR System for Discrimination between Pollen Parent and Seed Parent in Citrus

This study discusses the challenge of distinguishing between two high-quality mandarin cultivars, ‘Asumi’ and ‘Asuki’, which have been introduced and cultivated in Korea after being developed through crossbreeding in Japan. Owing to genetic similarities resulting from crossbreeding between the same parent cultivars, it is challenging to differentiate them morphologically at the seedling stage. This difficulty poses challenges for cultivation and harvesting on farms. To address this issue, we developed a method using sequence characteristic amplification region (SCAR) markers for rapid and accurate differentiation between the two cultivars. We selected specific primer sets from random amplified polymorphic DNA–SCAR combinations and sequence-related amplified polymorphism contrast markers. The multiplex PCR system using these molecular markers was able to identify 16 mandarin cultivars, including ‘Asumi’ and ‘Asuki’, among 30 cultivars. The use of these SCAR markers is expected to enhance citrus cultivation by accurately identifying mixed cultivars and facilitating proper harvest timing for citrus distribution. Additionally, the markers can help identify the genetic traits of hybrid varieties at the seedling stage.

Universal primer multiplex PCR assay for detection and genotyping of porcine astroviruses

Porcine astroviruses (PAstV) are members of the family Astroviridae, Mamastravirus genus and have been identified to have five genotypes (PAstV1–5). These viruses are highly prevalent in pigs and can cause enteric disease as well as neurological or respiratory symptoms depending on their genotypes. At present, the epidemiological impacts of some PAstV genotypes on pigs are largely unknown and hence continuously monitoring of these PAstVs may be needed. The purpose of this research was to develop an improved and efficient detection tool for PAstVs and to evaluate the developed method using clinical samples. Initially, a set of five chimeric primers (CP), each comprising genotype specific primer pairs with an identical universal adapter at the 5’ end, and a universal primer (UP) that is identical to universal adapter sequence, were designed. With these tools in place, a novel multiplex PCR system with universal primer was established for the simultaneous detection of the five types of PAstV. This method can specifically detect PAstV genotypes, with a limit of detection (LOD) of 5 copies/μL for each genotype irrespective of single or mixed target template. Using this new assay, 273 pig fecal samples were investigated for further assay evaluation. Among all samples, the positive rate was 70.0% with PAstV4 in 56.8% of the samples, PAstV2 in 38.8%, PAstV1 in 16.8%, and PAstV5 in 11.0%. More than one PAstV in a sample were detected in 39.2% of the samples. The consistency rate between the novel multiplex PCR and singleplex PCRs was 96.4–100%. Given its rapidity, specificity and sensitivity, the novel multiplex PCR is a useful approach for demonstrating single or mixed genotype infections of PAstV.

Development of a sensitive and user-friendly allele-specific PCR assay for adulteration detection of spices

Spice adulteration not only seriously interferes with their flavoring functions but also leads to life-threatening poisoning for consumers. To overcome the limitations of traditional methods in spice adulteration detection, a multiplex allele-specific PCR system was developed for molecular discrimination of four commonly used spices, Foeniculum vulgare Mill., Zanthoxylum bungeanum Maxim., Illicium verum Hook.f., and Syzygium aromaticum (L.) Merr. & L.M.Perry, from their corresponding adulterants based on chloroplast SNP markers. The developed assay, eliminating the obstacles of DNA sequencing and false negative results, can detect 0.1% of spice adulteration down to 0.01 ng level of genomic DNA with absolute allelic specificity and favorable efficiency. Based on the results, a standard operating procedure for using multiplex allele-specific PCR for spice adulteration detection was established. Therefore, the present study provided a simple, reliable, and sensitive molecular method for adulteration detection of spices.

The Development of a Multiplex PCR Assay for Fast and Cost-Effective Identification of the Five Most Significant Pathogenic Prototheca Species.

A multiplex PCR system (m-PCR) has been developed to accurately differentiate the five most important pathogenic Prototheca species, including the three species associated with infection in dairy cattle (P. ciferrii, P. blaschkeae, and P. bovis) and the two species associated with human infections (P. wickerhamii and P. cutis). The method is low-cost since it employs a simple "heat-shock" method in a TE buffer for DNA extraction. Furthermore, it requires only primers, a Taq polymerase, an agarose gel, and a molecular weight marker for identification. The method was based on published Prototheca cytochrome B sequences and was evaluated using reference strains from each of the five Prototheca species. The validity of the method was confirmed by identifying 50 strains isolated from milk samples. The specificity was tested in silico and with experimental PCR trials, showing no cross-reactions with other Prototheca species, as well as with bacteria, fungi, cows, algae, animals, or humans. The method could detect mixed infections involving two or three Prototheca species, providing a rapid test that delivers results within three hours.

Prevalence of Gene Rearrangement by Multiplex PCR in De Novo Acute Myeloid Leukemia in Adult Iraqi Patients.

Gene rearrangements of acute myeloid leukemia (AML) play a significant role in categorizing patients and provide valuable information about prognosis and treatment choices. However, in Iraq, the prevalence and prognostic significance of gene rearrangements in AML have not been previously examined. This study utilized a multiplex reverse transcription real-time PCR (RT-qPCR) system to identify gene rearrangements in a group of 115 adult patients from Iraq who had been diagnosed with De Novo AML. The diagnosis of AML was confirmed through blood film and flow cytometry. The ethical committee of the College of Medicine at the University of Baghdad provided approval for this research study. In this study, 66.1% of the patients diagnosed with acute myeloid leukemia (AML) exhibited distinct genetic abnormalities. Among these abnormalities, the most frequent was the rearrangement involving the KMT2A gene, observed in 19.9% of the patients. The risk stratification analysis revealed that 40% of the patients were classified as having a favorable risk, 4.3% as intermediate risk, and 25.2% as adverse risk. A subtype of AML known as core-binding factor (CBF) AML was identified in 21.7% of the cases, with 84% of these patients achieving complete remission. The NPM-RARA gene rearrangement, found in 43% of acute promyelocytic leukemia (APL) cases, was associated with a 71% complete remission rate. Among patients with KMT2A rearrangement, which accounted for 19.9% of all AML cases, the MLL-AF10 rearrangement was the most common, although only one patient with KMT2A rearrangement achieved complete remission. Furthermore, the analysis of demographic data revealed a significant association between increased risk and advanced age, presence of comorbidities, and FAB classification (M0 subtype). The prevalence of genetic rearrangements in Iraqi De Novo AML patients is higher than the global trend, highlighting the importance of genetic characterization in risk assessment and treatment decisions.

Optimization and Development of an Efficient 13 X-STRs Multiplex PCR System for Paternity Testing

X-chromosomal short tandem repeats (X-STRs) markers complement autosomal STR identification systems and valuable tools in complex kinship cases. Objective: To develop a multiplex PCR system that consists of 13 X-chromosome STR markers, including GATA172D05, DXS8378, DXS6801, DXS6793, DXS6810, DXS7132, GATA31E08, DXS9902, HPRTB, DXS6789, DXS7423, DXS8377, DXS981 and sex-determining locus Amelogenin. Methods: Primer sequences of all X-STR markers were acquired from the Genome databases, and the original sequences for HPRTB, DXS6789, DXS7423, DXS8377 and DXS981 were modified to eliminate primer-dimer formation and optimize melting temperatures to increase annealing efficiency. All primer pairs were labelled with fluorescent dyes to support amplification in a multiplex PCR, and the cycling conditions for multiplex PCR were optimized. Alleles for each locus were bi-directionally sequenced to determine the exact repeat size, and alleles generated in multiplex reactions were undistinguishable from alleles produced in a single marker PCR reaction. Results: The combined power of discrimination of 13 X-STRs was 2.96 x 10-13 and 2.58 x10-8 in females and males, respectively. Conclusions: In conclusion, we have developed a 14-plex PCR system that can potentially be used for parentage testing and forensic casework studies.

Monitoring the growth dynamics of Tetragenococcus halophilus strains in lupine moromi fermentation using a multiplex-PCR system

ObjectiveThe microbiota of a seasoning sauce fermentation process is usually complex and includes multiple species and even various strains of one species. Moreover, composition and cell numbers of individual strains vary over the course of the entire fermentation. This study demonstrates the applicability of a multiplex PCR system to monitor growth dynamics of Tetragenococcus (T.) halophilus strains in order to evaluate their performance and help to select the most competitive starter strain.ResultsIn a previous study we isolated T. halophilus strains from multiple lupine moromi fermentation processes and characterized them. In this study we wanted to monitor the growth dynamics of these strains in a competitive lupine moromi model fermentation process using a multiplex PCR system. Therefore, pasteurized lupine koji was inoculated with eight different T. halophilus strains, six from lupine moromi, one from an experimental buckwheat moromi fermentation process and the type strain DSM 20,339T, to create the inoculated lupine moromi pilot scale fermentation process. With the multiplex PCR system, we could detect that all strains could grow in lupine moromi but, that TMW 2.2254 and TMW 2.2264 outperformed all other strains. Both strains dominated the fermentation after three weeks with cell counts between 4 × 106 to 4 × 107 CFU/mL for TMW 2.2254 and 1 × 107 to 5 × 107 CFU/mL for TMW 2.2264. The pH dropped to value below 5 within the first 7 days, the selection of these strains might be related to their acid tolerance.

Studying the Genetic Diversity of the Rice Break Cause <i>Pyricularia oryzae</i> Cav. on the Complex of Molecular and Morphological Features

On the basis of molecular genetic approaches, the study of the biodiversity of the phytopathogenic fungus Pyricularia oryzae Cav. in the south of Russia and a classification of the morphological and cultural characteristics of the pathogen was carried out. For 2020–2022 monitoring was carried out and 24 strains of the pathogen were isolated from the affected herbarium material collected from the fields of five ecological rice growing zones of the Krasnodar Territory (Krasnoarmeisky, Kalininsky, Abinsky, Slavyansky districts, Krasnodar). Using the multiplex PCR system developed by us based on fragment analysis, five genotypes were identified among the studied Pyricularia oryzae Cav. strains over the entire period of the project, each of which is characterized by a unique genetic profile. Compiled their “DNA passport”. The studied isolates of the causative agent of blast were characterized by morphological and cultural characteristics. Six morphotypes of the pathogen have been identified.