PCR-restriction Fragment Length Polymorphism Analysis Research Articles

Identification of Fish Species Involved with Ciguatera Food Poisoning in Okinawan Waters by Using PCR-RFLP analysis

Ciguatera fish poisoning (CFP), known as a seafood-borne disease, is caused by consumption of fish contaminated with ciguatoxins in tropical and subtropical sea. The ciguatera fishes, Variola louti, Lutjanus monostigma and L. bohar have an absolute majority in the Ryukyu Archipelago, southwestern Japan. We developed the cluster analysis of phylogenetic tree by using mitochondrial (mt) DNA 16S rRNA sequences of V. louti, L. monostigma and L. bohar and differentiate them from morphologically similar species (L. fulviflamma, L. russellii, L. argentimaculatus, Plectropomus leopardus and V. albimarginata) in our previous study. The fish were acquired from the coastal waters of the Ryukyu Archipelago, and a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) marker of the mtDNA 16S rRNA region was used, employing the restriction enzymes BmgT120 I, Dde I, and SnaB I, to identify the fish species responsible for CFP. These results showed that a PCR-RFLP marker can be obtained more easily than a nucleotide sequence.

Zoonotic human liver flukes, a type 1 biocarcinogen, in freshwater fishes: genetic analysis and confirmation of molluscan vectors and reservoir hosts in Bangladesh

BackgroundOpisthorchiid flukes, particularly Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, and Metorchis spp. are the most common fish-borne zoonotic human liver flukes (hLFs). Liver fluke infections are more prevalent in resource-deprived and underprivileged areas. We herein estimated the prevalence of the metacercariae (MC) of major hLFs in common large freshwater fishes (lFWF) marketed for human consumption from some selected areas of Bangladesh along with detection of their molluscan vectors and reservoirs.MethodsThe current status of fish-borne zoonotic hLF infections in lFWF was investigated along with their molluscan vectors and mammalian reservoir hosts in Mymensingh and Kishoreganj in Bangladesh from July 2018–June 2022 using conventional and multiple molecular techniques, such as PCR, PCR-restriction fragment length polymorphism (RFLP), sequencing, and bioinformatic analyses. The infection rate of fishes was analyzed using the Z-test and the loads of MC were compared using the chi-squared (χ2) test.ResultsThe MC of C. sinensis, Opisthorchis spp., and Metorchis spp. were detected in 11 species of common and popular lFWF. In lFWF, the estimated prevalence was 18.7% and the mean load was 137.4 ± 149.8 MC per 100 g of fish. The prevalence was the highest (P < 0.05) in spotted snakehead fishes (Channa punctata, 63.6%). The highest rate of infection (P < 0.05) was observed with the MC of C. sinensis (11.8%). Metacercariae were almost equally (P > 0.05) distributed between the head and body of fishes. The infection rate was slightly higher in cultured (19.6%) fishes. The MC of C. sinensis, O. felineus, O. viverrini, and Metorchis orientalis in fishes were confirmed using PCR, PCR-RFLP and bioinformatics. The cercariae of opisthorchiid (Pleurolophocercus cercariae) flukes were only recovered from Bithynia spp. (3.9%, 42 out of 1089). The ova of hLFs from dogs (4.3%, 5 out of 116) and cats (6.0%, 6 out of 100), and adult flukes (M. orientalis) from ducks (41.1% 113 out of 275) were detected.ConclusionsThe MC of hLFs are highly prevalent in fresh water fishes in Bangladesh. Reservoir hosts, such as street dogs, cats, and ducks carried the patent infection, and residents of Bangladesh are at risk.Graphical

Interleukin 17F Gene Polymorphism as a Potential Protective Factor in the Immunopathology of Ocular Toxoplasmosis.

Ocular toxoplasmosis (OT) is characterised by intraocular inflammation due to Toxoplasma gondii infection. Studies have found that interleukin 17 (IL-17) plays a central role in the pathology of OT. However, nucleotide variability in IL17 and interleukin 17 receptor (IL17R) genes has not been characterised in OT. As cytokine gene polymorphisms may influence the expression of these molecules, the aim of this study was to verify whether IL17A (rs2275913), IL17F (rs763780), IL17RA (rs4819554) and IL17RC (rs708567) polymorphisms are associated with OT in a Brazilian population. This study enrolled 214 patients seropositive for T. gondii (110 with OT and 104 without) and 107 controls. Polymorphisms were identified by PCR-restriction fragment length polymorphism analysis, validated by DNA sequencing with chi-square and multivariate analyses being used to assess possible associations between polymorphisms and OT. Logistic regression under the dominant model revealed a protection factor against OT of the C mutant allele of the IL17F (rs763780) polymorphism. The T/C-C/C genotypes were significantly more common in patients without OT compared to those with OT (p value = 0.0066) and controls (p value = 0.014). Findings from this study suggest that the IL17F polymorphism may have an influence in the immunopathology of OT in Brazilian individuals.

A PCR-RFLP Analysis for the Diagnosis of DAS and T-2 Genotypes in Fusarium goolgardi

Fusarium pathogens and their mycotoxins are considered as the main threats to cereal production and food safety worldwide. However, due to the constant discovery of new Fusarium species especially along with mycotoxin production profile differentiation in certain species, efforts on their species composition, geographical distribution, and chemotype proportion are urgently required. In the Fusarium goolgardi species, two distinct trichothecene (TB) genotype populations have recently been identified. Previous studies have shown that the structural variance of TBs biosynthesized by the two genotypes is attributed to the Tri1 gene. Polymorphisms of Tri1 gene from type A TB-producers were investigated in different Fusarium species in the current study. According to these DNA sequence variations identified in Tri1 gene sequences, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) based diagnostic approach for the differentiation of T-2 and 4,15-diacetoxyscirpenol (DAS) genotypes in F. goolgardi was successfully developed. The PCR-RFLP assay will facilitate the studies on geographic distribution, frequency and other aspects of the two genotypes within F. goolgardi species.

Correlation of DNA load, genotyping, and clinical phenotype of Mycoplasma pneumoniae infection in children.

This study aimed to investigate the correlation between Mycoplasma pneumoniae (MP)-DNA load in the bronchoalveolar lavage fluid (BALF) of children with MP pneumonia (MPP) and its subtypes, relevant laboratory data, imaging, extrapulmonary complications in infected children, and its clinical significance in evaluating the disease. Children hospitalized with MPP at Tianjin Children's Hospital between December 2017 and December 2020 were selected for the study, excluding those with mixed viral, bacterial, and fungal infections. Children were divided into low- and high-load groups according to the MP DNA load in BALF using real-time quantitative fluorescence polymerase chain reaction (PCR). After a successful MP culture, positive specimens were subjected to PCR-Restriction fragment length polymorphism and Multiple-locus variable number tandem repeat analysis typing. Basic data, clinical information, laboratory data, and radiological results were collected from all children included in the study. The PI-I type dominated the different load groups. Children in the low-load group had more wheezing and shortness of breath; however, children in the high-load group had a higher length of hospitalization, maximum fever temperature, higher chills/chilliness, incidence of abdominal pain, and higher C-reactive protein (CRP), procalcitonin (PCT) and aspartate aminotransferase (AST) levels. Children in the high-load group were more likely to have imaging changes such as pleural effusion, and the incidence of respiratory infections and extrapulmonary complications was higher than that of those in the low-load group. We applied Spearman's correlation analysis to clarify the relationship between MP DNA load and the clinical severity of MPP. We found that MP DNA load was positively correlated with length of hospitalization, maximum fever temperature, CRP, PCT, Interleukin-6 (IL-6), and AST levels, and negatively correlated with fever and cough durations, white blood cell count (WBC), and proportion of monocytes (MONO). The degree of correlation was as follows: length of hospitalization > IL-6 > cough duration > AST > fever duration > PCT > WBC > proportion of MONO > maximum fever temperature > CRP levels. MP DNA load was not correlated with MP typing but was significantly correlated with the children's clinical phenotype. Therefore, the MP DNA load helps in the early diagnosis of infection and can better predict disease regression.

Phylogenetic Variation of Tri1 Gene and Development of PCR-RFLP Analysis for the Identification of NX Genotypes in Fusarium graminearum Species Complex.

NX toxins have been described as a novel group of type A trichothecenes produced by members of the Fusarium graminearum species complex (FGSC). Differences in structure between NX toxins and the common type B trichothecenes arise from functional variation in the trichothecene biosynthetic enzyme Tri1 in the FGSC. The identified highly conserved changes in the Tri1 gene can be used to develop specific PCR-based assays to identify the NX-producing strains. In this study, the sequences of the Tri1 gene from type B trichothecene- and NX-producing strains were analyzed to identify DNA polymorphisms between the two different kinds of trichothecene producers. Four sets of Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were successfully developed to distinguish the common type B trichothecene producers and NX producers within FGSC. These promising diagnostic methods can be used for high-throughput genotype detection of Fusarium strains as a step forward for crop disease management and mycotoxin control in agriculture. Additionally, it was found that the Tri1 gene phylogeny differs from the species phylogeny, which is consistent with the previous studies.

Detection of genetic variations in the GDF9 and BMP15 genes in Kazakh meat-wool sheep.

Kazakh meat-wool sheep are of great interest because of the intrabreed multifetal type's high productivity of 140 %-160 %. Genes encoding growth differentiation factor-9 (GDF9) and bone morphogenetic protein 15 (BMP15) are promising candidates for studying sheep productivity, as they affect fertility in mammals, including sheep. Thus, the purpose of this study was to assess the fertility of the Kazakh meat-wool sheep breed based on GDF9 and BMP15 candidate genes of fecundity for the selection of animals with valuable genotypes. We selected 300 heads of the Kazakh meat-wool sheep breed from two populations for PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis, 15 of which were subsequently used for sequencing of exon regions of the GDF9 and BMP15 genes. The sheep populations were tested for G1 and G8 mutations of the GDF9 gene and B2 and B4 mutations of the BMP15 gene. The PCR-RFLP analysis revealed that 59 (19.7 %) of the 300 Kazakh meat-wool breed sheep were heterozygous carriers of the G1 mutation (genotype AG) of the GDF9 gene, and sequencing analysis supported these results. The comparative phylogenetic analysis showed a clear separation of Kazakh meat-wool sheep wild types and carriers of the G1 mutation. This mutation was reported to have a relationship with the animals' litter size in other sheep breeds. For this reason, similar relationships should be investigated in Kazakh meat-wool sheep. However, G8, B2, and B4 mutations were not detected among the studied animal populations, showing that these mutations are not characteristic of the Kazakh meat-wool sheep breed.

Prevalence of 4a/4b polymorphic variants of the eNOS gene introne in patients with different types of encephalopathies

Questions related to the disruption of vasoregulatory processes as essential factor in neurological pathology require further study. The leading role in the vasoregulatory mechanisms is played by endothelial NO synthase which gene has 15 allelic variants. Recent data indicated a probable association between eNOS gene polymorphism and cerebrovascular diseases. The aim of the present work was to study the prevalence of the 4a/4b introne polymorphism of the eNOS gene in patients with various types of encephalopathies and to evaluate the influence of a particular genotype of the studied gene on the occurrence and/or progression of encephalopathy. A total of 96 patients with encephalopathies of various genesis: chronic traumatic encephalopathy, chronic alcohol-induced encephalopathy, chronic vascular encephalopathy, post-infectious encephalopathy were involved in the study. The patients received inpatient treatment in the neurological department of “Ternopil Regional Clinical Psychoneurological Hospital”. Molecular and genetic differentiation of the studied gene variants was carried out by allele-specific PCR or PCR-restriction fragment length polymorphism analysis by standard operational protocols. Analysis of the frequency distribution of eNOS gene 4a/4b polymorphic variant showed that 4b allele prevailed among patients with all types of encephalopathies carriers­. Relative to practically healthy individuals the difference was found only in patients with chronic vascular encephalopathy (CVE), among whom about 39% were carriers of the 4a allele. The presence of the 4a allele was shown to increased the risk of CVE occurrence and/or progression by 4.5 times. The results obtained suggest the reasonability to include the 4a/b intron polymorphism of the eNOS gene in a genetic panel to monitor patients CVE. Keywords: 4a/4b introne, encephalopathies, endothelial NO-synthase, eNOS gene polymorphism

Circulating miRNAs and their functional genetic variants in pseudoexfoliative glaucoma: potential of miR-146a-5p as a diagnostic biomarker.

The etiology and pathogenesis of pseudoexfoliation syndrome (PEX) and its advancement into pseudoexfoliative glaucoma (PEG) are not fully understood. In this study, we aimed to evaluate the possible role played by two circulating microRNAs (miR-146a-5p and miR-196a-5p) in plasma and their functional genetic variants MIR146A rs2910164 and MIR196A2 rs11614913 in susceptibility to PEG or PEX. Plasma miRNA relative expression of 27 patients with PEG, 25 patients with PEX and 27 controls was determined using quantitative RT-PCR, and fold change was calculated using the 2-ΔΔCt method. Genotyping of 300 patients with PEG, 300 patients with PEX, and 300 controls was performed using a PCR-restriction fragment length polymorphism analysis. Plasma miR-146a-5p relative expression was significantly elevated in patients with PEG (3.9-fold) (P < .000) and patients with PEX (2.7-fold) relative to controls (P = .001). The diagnostic ability of plasma miR-146a-5p expression fold change was good for discriminating PEG vs. controls (AUC = 0.897, P < .000), and the optimal decision threshold was 1.83 (sensitivity = 74%, specificity = 93%). Plasma miR-196a-5p relative expression did not differ significantly between study groups. No significant difference in terms of the minor allele frequency or the distribution of genotypes for MIR146A rs2910164 G/C or MIR196A2 rs11614913 C/T was observed between study groups. Circulating miR-146a-5p can contribute to the risk of PEX/PEG. Therefore, we propose that plasma miR-146a-5p can be developed as a potential biomarker for the minimally invasive diagnoses of PEX/PEG and as a potential therapeutic target with further studies.

Molecular characterization of multidrug resistant (MDR) clinical isolates of Pseudomonas aeruginosa from Nsukka, South Eastern Nigeria.

P. aeruginosa is an important nosocomial pathogen with increasing resistance to antibiotics. Objective: This study was performed to evaluate the genetic relatedness of MDR clinical isolates of P. aeruginosa. A total of 1000 samples were analysed in the study. Antibiotic resistance profiles of the isolates were determined using Kirby Bauer disk diffusion method. Polymerase chain reaction (PCR) and sequencing were simultaneously used to detect the consensus region of 16S rRNA. Genetic relatedness of the isolates was determined using restriction patterns from ALU 1 digest and random amplified polymorphic DNA. Out of the 192 P. aeruginosa isolates recovered, 136 (78.83%) were multidrug resistant. Sequence analysis of the confirmed isolates (80.68%) revealed that all the isolates shared homology with each other and also showed sequence similarity to known strains of P. aeruginosa (ATCC 27853; KT 315654; KU 321274 and KT894767). The PCR-Restriction fragment length polymorphism (PCR-RFLP) analysis revealed that there was a lot of genetic relatedness among the isolates. The RFLP finger printing technique detected seven distinct RFLP types among the isolates. Thus, study shows that there is high prevalence of MDRPA and high degree of genetic relatedness among the MDRPA isolates circulating in Nsukka area.

Diagnostic difficulties in non-tuberculous mycobacterial infection in lung transplant recipients.

Despite antimicrobial prophylaxis, 34% to 59% of lung transplant recipients experience severe life-threatening opportunistic infections, sometimes caused by Nontuberculous Mycobacteria (NTM) and Nocardia. Although differentiating these infections is of utmost importance for effective treatment, it can be challenging as they share morphological and growth characteristics. Therefore, culture remains the gold standard for laboratory confirmation. With the aid of novel molecular methods performed on the cultured organisms, diagnosis may be accomplished rapidly and precisely. We present a case of a lung transplant recipient with a pulmonary infection where long, thin, beaded, branching filamentous organisms were seen with Acid-Fast Bacilli (AFB) and Modified Gomori's Methenamine Silver (GMS) stains in bronchoalveolar lavage sample. Cytological characteristics led to the suspicion of a Nocardia species infection. However, culture and the PCR-restriction fragment length polymorphism analysis (PRA) identified M. fortuitum. Additionally, antibiotic resistance was detected, which aided in choosing the appropriate treatment. Therefore, to overcome such diagnostic difficulties to differentiate NTM and Nocardia, a multidisciplinary approach including culture, molecular methods, and cytology is needed to enhance clinical outcomes.

PCR-RFLP Based Detection of atypical Mycobacteria Isolated from Aquatic Environment

Non-tuberculous mycobacteria (NTM) serve as the causative agent for fish tuberculosis which is a chronic progressive disease covering a vast range of cultured and wild, freshwater and marine environments in different climatic conditions. Detection of environmental Mycobacteria in a conventional way is generally dependent on the basis of phenotypic, histopathological and biochemical characterization. The present study focused on the distribution and occurrence of mycobacterial pathogens in clinically infected fishes of a variety of species, water and soils from different aquaculture sources in West Bengal, India. Samples were collected from four distinct regions where fishes were examined for granulomatous inflammation. A total of 161 fishes, 127 water and 97 soil samples were examined and 132 (81.98 %), 65 (51.18 %) and 36 (37.11 %) appeared as acid-fast positive respectively. Presences of acid-fast bacteria within necrotic tissue of infected fishes were detected by Zn staining. Morphology, biochemical characteristics and 16S rRNA gene sequencing of bacteria were satisfactory to show the acid-fast bacilli belong to the Genus Mycobacteria. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was carried out to analyze the rpoB gene sequencing to confirm Mycobacteria down to the species level. Ten different NTM species were identified using PCR-RFLP analysis. Mycobacterium fortuitum type I was the most abundantly isolated species among all the sample sources. The present study suggests that water play a major role in the epidemiology of environmental Mycobacteria, infecting aquatic organisms and conducting potential public health hazard, especially those who are immune-compromised individual.

Genetic diversity of Plasmodium vivax populations from the China–Myanmar border identified by genotyping merozoite surface protein markers

BackgroundParasite diversity and population structure influence malaria control measures. Malaria transmission at international borders affects indigenous residents and migrants, defying management efforts and resulting in malaria re-introduction. Here we aimed to determine the extent and distribution of genetic variations in Plasmodium vivax populations and the complexity of infections along the China–Myanmar border.MethodsWe collected clinical P. vivax samples from local and migrant malaria patients from Laiza and Myitsone, Kachin State, Myanmar, respectively. We characterized the polymorphisms in two P. vivax merozoite surface protein markers, Pvmsp-3α and Pvmsp-3β, by PCR-restriction fragment length polymorphism (PCR–RFLP) analysis. We sought to determine whether these genetic markers could differentiate these two neighboring parasite populations.ResultsPCR revealed three major size variants for Pvmsp-3α and four for Pvmsp-3β among the 370 and 378 samples, respectively. PCR–RFLP resolved 26 fragment-size alleles by digesting Pvmsp-3α with Alu I and Hha I and 28 alleles by digesting Pvmsp-3β with Pst I. PCR–RFLP analysis of Pvmsp-3α found that infections in migrant laborers from Myitsone bore more alleles than did infections in residents of Laiza, while such difference was not evident from genotyping Pvmsp-3β. Infections originating from these two places contained distinct but overlapping subpopulations of P. vivax. Infections from Myitsone had a higher multiplicity of infection as judged by the size of the Pvmsp-3α amplicons and alleles after Alu I/Hha I digestion.ConclusionsMigrant laborers from Myitsone and indigenous residents from Laiza harbored overlapping but genetically distinct P. vivax parasite populations. The results suggested a more diverse P. vivax population in Myitsone than in the border town of Laiza. PCR–RFLP of Pvmsp-3α offers a convenient method to determine the complexity of P. vivax infections and differentiate parasite populations.

Eight single nucleotide polymorphisms and their association with food habit domestication traits and growth traits in largemouth bass fry (Micropterus salmoides) based on PCR-RFLP method.

The largemouth bass (Micropterus salmoides), an economically important freshwater fish species widely farmed in China, is traditionally cultured using a diet of forage fish. However, given the global decline in forage fish fisheries and increasing rates of waterbody pollution and disease outbreaks during traditional culturing, there is a growing trend of replacing forage fish with formulated feed in the largemouth bass breeding industry. The specific molecular mechanisms associated with such dietary transition in this fish are, nevertheless, poorly understood. To identify single nucleotide polymorphisms (SNPs) related to food habit domestication traits and growth traits in largemouth bass fry, we initially genotyped fry using eight candidate SNPs based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, with genetic parameters being determined using Popgen32 and Cervus 3.0. Subsequently, we assessed the associations between food habit domestication traits of largemouth bass fry and these SNPs using the Chi-square test or Fisher's exact test. Furthermore, we used a general linear model to assess the relationships between the growth traits of largemouth bass fry and these SNPs. The Pearson correlation coefficient between growth traits and the SNPs was also determined using bivariate correlation analysis in IBM SPSS Statistics 22. Finally, the phenotypic variation explained (PVE) by the SNPs was calculated by regression analysis in Microsoft Excel. The genotyping results obtained based on PCR-RFLP analysis were consistent with those of direct sequencing. Five SNPs (SNP01, SNP02, SNP04, SNP05, and SNP06) were found to be significantly correlated with the food habit domestication traits of fry (P < 0.05); SNP01 (P = 0.0011) and SNP04 (P = 0.0055) particularly, had showed highly significant associations. With respect to growth traits, we detected significant correlations with the two SNPs (SNP01 and SNP07) (P < 0.05), with SNP01 being significantly correlated with body length, and height (P < 0.05), and SNP07 being significantly correlated with body height only (P < 0.05). Our findings indicated that the PCR-RFLP can be used as a low-cost genotyping method to identify SNPs related to food habit domestication and growth traits in largemouth bass, and that these trait-related SNPs might provide a molecular basis for the future breeding of new varieties of largemouth bass.

Frequency and genotyping of Giardia duodenalis in dogs of Urmia, northwest of Iran.

Giardia duodenalis is a zoonotic protozoan infecting various vertebrates such as humans and domestic animals. The aim of this study was to determine the frequency and genotypes of G. duodenalis using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in dogs of Urmia, Iran. Overall, 246 stool specimens were collected from 100 pet, 49 stray, and 97 shelter dogs in the Urmia, Iran. Totally, seven samples (2.48%) were microscopically positive in terms of Giardia cyst. The PCR-RFLP analysis revealed that three (1.21%) and two (0.83%) samples have the C and D genotypes, respectively. In addition, two samples (0.83%) were belonged to the AI sub-group. A significant association was determined between the frequency of Giardia infection and life style, age, and stool form of dogs. The findings of the study showed the high frequency of Giardia infection in stray dogs and the dogs under one-year-old. Furthermore, the C and D genotypes of G. duodenalis were predominant in dogs of Urmia, Iran.

Molecular detection and dense granule antigen 6 genotyping of feline Toxoplasma gondii in Phayao, Thailand.

Globally, toxoplasmosis is an important zoonotic parasite infection of many warm-blooded animals (including humans). Toxoplasma gondii oocysts are widespread, and their contamination can be primarily attributed to the members of the Felidae family. This study aimed to estimate the prevalence and determine the dense granule antigen 6 (GRA6) genotype of T. gondii among domestic cats in the Phayao Province, Thailand. A total of 124 fecal samples were collected from owned cats in the Muang district, Phayao, Thailand, from January to December 2020. Fecal samples were tested for the presence of T. gondii DNA using targeted B1 gene polymerase chain reaction (PCR) amplification, and positive samples were subsequently analyzed for their T. gondii genotype through PCR-restriction fragment length polymorphism (RFLP) analysis and sequencing of the GRA6 gene. Among the 124 samples, 46 (37.1%) were tested positive for T. gondii. Only 10 positive DNA samples were successfully amplified for the GRA6 marker. Subsequent PCR-RFLP and sequence analyses indicated that all T. gondii isolates from cats in Phayao belonged to GRA6 genotype I. Data revealed that toxoplasmosis is remarkably distributed among (studied) domestic cats in Phayao, Thailand. Moreover, the virulent GRA6 allele was found to be circulated among domestic cats in this area. However, no significant correlation was observed between infection rates and different risk factors, which indicated that pet cats of any age, gender, or breed have similar risks of being infected with T. gondii. Our results further suggested that infective oocysts of T. gondii are widely distributed and that environmental contamination with these oocysts will introduce more risks of disease transmission to humans and other animals.

Time and cost-efficient identification of Trichophyton indotineae.

During the past 5 years, an outbreak of recalcitrant dermatophytosis due to a novel Trichophyton species generally resistant to terbinafine, T.indotineae, has spread out from South Asia to many countries around the World. These isolates cannot be reliably differentiated from other Trichophyton spp. on the basis of morphological traits and the current laboratory diagnostics relies on sequencing of ribosomal DNA ITS region. In this study, we aimed to introduce two inexpensive and rapid PCR-based assays for differentiation between T.indotineae and other dermatophytes. The first introduced assay is based on PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, involving the amplification of TOP2 sequences and the digestion of PCR products by Cfr13I restriction enzyme. The second assay is proposed as conventional endpoint species-specific PCR amplification of the C120-287 intergenic locus. To validate the assays, a total of 191 Trichophyton spp. and 2 Microsporum canis strains with known ITS region sequences were used. From the T.mentagrophytes / T.interdigitale species complex (TMTISC), strains with 18 different ITS genotypes were tested. The sample of TMTISC isolates included 41 T.indotineae strains. TOP2 PCR-RFLP and T.indotineae-specific PCR were positive with testing on DNA of all 41 T.indotineae isolates and two strains of T.mentagrophytes belonging to ITS Types XIII and XVI, but negative with other species and other TMTISC ITS genotypes (n=152). Therefore, the specificity of both new assays was 99%. The two developed diagnostic assays provide accurate and cost-effective means of identifying cultured T.indotineae isolates.

GENOTYPING OF NATIVE AND IMMUNOLOGICALLY CASTRATED GILTS BY QTL-MARKER OF RYR1 AND CTSD

In the study conducted, the allele effect of the SNP studied was determined (Single Nucleotide Polymorphism) ryanodine receptor gene 1 RYR1 (g.1843 C&gt;T) and cathepsin gene D CTSD (g.70 G&gt;A) for fattening performance of the control (native) and experimental (immunologically castrated) groups of gilts the final hybrid (Large White × Landrace) × Maxgro. It was held a population analysis of polymorphism using software «GenAlEX6» to find out if the allele effect of the investigated is different (SNP) on the productivity of hybrid gilts - of the native in the number of samples (n=38) and immunologically castrated (n=30) grown in conditions of LLC SPE "Globinsky Pig Farm". Laboratory tests are carried out in the laboratory of genetics the basis of the Institute of Pig Breeding and Agricultural Production NAAS of Ukraine. Due to the fact that information on polymorphism according to QTL markers of ryanodine receptor 1 and cathepsin D in the population of immunologically castrated pigs that are bred in Ukraine is absent requires an experiment to determine which alleles will be determined as desirable in marker breeding of pigs under conditions of immunological castration. Polymorphism of the studied gene RYR1 (g.1843 C&gt;T) and CTSD (g.70 G&gt;A) was determined by the method of PCR-RFLP (Polymerase chain reaction-restriction fragment length polymorphism) analysis. Researched by QTL (quantitative trait loci) ryanodine receptor gene 1 RYR1 (g.1843 C&gt;T) - is a marker gene whose polymorphism is associated with the quality of meat, and is responsible for the developed muscles of the carcass and limbs as well as pigs' tendency to stress (PSS) – RYR1TT. In the SNP, the RYR1 allele T frequency was 0.941 and is higher than the allele frequency C of 0.059. The genotype RYR1СС was found in only one native pig with (ADG=0.677g/163day). The genotype RYR1TT with a frequency of 0.94 genotypes prevails RYR1СС =0.06. Gilts with genotype RYR1TT are stress-sensitive. Indicator (ADG) for immunologically castrated gilts is (ADG=0.875g/149day) with a significant difference with uncastrated pigs in indicator of – average growth rate 156days. The PIC level for locus RYR1 is low at 0.10, which limits its use for associated studies. No pigs with the RYR1CC genotype were found among the research team. However, it was found only in 1 head of female pigs – the control group as a result of a single case in this population. Animals with the genotype RYR1TT 7 days earlier reached a live weight of 100 kg from the control group. The gene of cathepsin D CTSD (g.70 G&gt;A) – is associated with precocity and efficiency of feed use. The breed character of a frequency distribution of gene alleles was established CTSD (g.70 G&gt;A) was 0.595 by G allele frequency significantly prevailing allele A 0.405 with the index of the information content of PIC loci at the optimal level of 0.37. A research group of pigs with monomorphic genotype CTSDGG prevailed the control group (ADG=0.770g/159day) by age reaching a live weight of 100 kg in 151 days with an average daily increase of 0.808g. A similar situation was formed as a result of the average daily increase in experimental pigs with genotype CTSDGА 0.879 g. with the same age, reaching a live weight of 100 kg in 147 days (ADG=0.858g; 0.879/147day) and the average growth rate other than uncastrated gilts. Hybrid pigs with genotype CTSDGА=0.85 genotypes prevail CTSDGG with a frequency of 0.19.

Molecular authentication of the medicinal crop Portulaca oleracea and discrimination from its adulterants in herbal markets using PCR-restriction fragment length polymorphism (PCR-RFLP) analysis

Portulaca oleracea (PO) is a popular traditional Chinese medicine (TCM) used to cool the body and reduce toxins. Authentication of Chinese herbal medicines (CHM), such as medicinal crop PO, is important and is a key step for quality control during TCM production. Adulteration of CHM reduces the efficacy of TCM and can lead to side effects in patients. Therefore, it is urgent to develop a reliable method for the verification of CHM in the TCM market. In this study, a DNA molecular method was established for rapid authentication of PO. Herein, four molecular markers, ITS1, ITS2, rbcL, and trnL, were chosen for evaluation of the sequence similarity between PO and its frequently used adulterants. Based on the phylogenetic analysis results, ITS2 was selected as the most promising molecular marker for PO authentication. Furthermore, PCR-amplified ITS2 was used to establish an RFLP pattern to distinguish PO from its adulterants. The specific restriction enzyme HpyF3I was found to quickly and effectively distinguish PO from the commonly confused plant Bacopa monnieri (BM) and other adulterants. In conclusion, this DNA molecular method can be used for PO authentication and be practically applied to audit PO plants on the herbal market and in the field.

Comparison of culture and polymerase chain reaction-restriction fragment length polymorphism for identification of various Capnocytophaga species from subgingival plaque samples of healthy and periodontally diseased individuals.

Introduction:Capnocytophaga are facultative anaerobic Gram-negative bacilli and recognized as opportunistic pathogens of various extraoral infections. Only a few studies attempted to identify all the seven species of Capnocytophaga phenotypically and genotypically in healthy individuals and patients with chronic periodontitis. Studies to determine the prevalence of Capnocytophaga in subgingival plaque samples from healthy individuals, chronic gingivitis and periodontitis among Indian population are lacking.Aim:The aim of this study was to identify and compare the presence of Capnocytophaga species phenotypically through microbial culture and biochemical tests and genotypically through polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) in subgingival plaque of healthy individuals and patients with chronic gingivitis and chronic periodontitis.Materials and Methods:A total of 300 subjects, 100 each with gingivitis, periodontitis and periodontally healthy gingiva subjected, were included. Subgingival plaque was collected and was cultured for phenotypic identification (microbial culture and biochemical test), and for genotypic identification, DNA extraction was done and PCR-RFLP analysis was performed to identify the genus Capnocytophaga and also to identify different species of Capnocytophaga.Results:Of 300 individuals, Capnocytophaga species were identified from 237 (79%) individuals by PCR and 82 (27.33%) by culture. The prevalence of Capnocytophaga ochracea was found to be higher with both the methods followed by Capnocytophaga gingivalis and Capnocytophaga granulosa. Capnocytophaga genospecies, Capnocytophaga leadbetteri and Capnocytophaga Sputigena were isolated only by culture with very low prevalence that is 1.33%, 1.33% and 0.66%, respectively. We could not get any isolate of Capnocytophaga haemolytica by any of the two methods.Conclusion:Capnocytophaga species could be found in gingival sulci as well as periodontal pockets and can be detected by culture and PCR-RFLP. However, higher prevalence of these species in healthy compared to disease requires further analysis to determine their role in healthy and diseased periodontium.