scholarly journals Molecular Identification and Characterisation of Aspergillus Flavus Isolates Originating from Serbian Wheat Grains

2020 ◽  
Vol 49 (4) ◽  
pp. 382-389
Author(s):  
J. Krulj ◽  
N. Ćurčıć ◽  
A. Bočarov Stančıć ◽  
J. Kojıć ◽  
L. Pezo ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Molecular Genetic ◽  
Pcr Amplification ◽  
Its Region ◽  
Holistic Approach ◽  
Accurate Identification ◽  
Essential Point ◽  
Climate Conditions ◽  
Wheat Grains ◽  
Pcr Rflp

During previous years, regarding the shifts in climate conditions in temperate region, such as occurrence of high temperatures and prolonged drought, increased occurrence frequencies of Aspergillus flavus and aflatoxins in cereal grains were recorded. A reliable and accurate identification of the fungi is of great importance for evaluating the microbiological risks of contamination. The essential point of the present investigation was molecular characterisation and identification of A. flavus isolates originating from common wheat and spelt grains collected after harvest during the period of three years (2015–2017) in Northern Serbia. A holistic approach that included PCR amplification of two DNA genomic regions and PCR-RFLP assay followed by fragment length analysis, provided complete and comprehensive characterisation of A. flavus isolated from wheat grains. The presented results indicate that there was no difference among the tested Aspergillus isolates on the molecular–genetic level. All 38 strains were identified as A. flavus by sequencing of combined ITS region and β-tubulin gene fragments (acc. no.: MH582473 to MH582510). PCR-RFLP method in combination with a Lab-on-a-chip (LoaC) electrophoresis can be successfully used to rapidly identify A. flavus isolates.

scholarly journals PCR-RFLP of 16S ribosomal DNA to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples

2010 ◽  
Vol 43 (1) ◽  
pp. 100-101 ◽  
Author(s):  
Aline Weber Medeiros ◽  
Pedro d'Azevedo ◽  
Rebeca Inhoque Pereira ◽  
Ana Paula Cassenego ◽  
Sueli Van Der Sand ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Food Samples ◽  
Correct Identification ◽  
Accurate Identification ◽  
16S Ribosomal Dna ◽  
Pcr Rflp ◽  
Enterococcus Casseliflavus ◽  
Dna Pattern ◽  
Enterococcus Gallinarum ◽  
Rdna Analysis

INTRODUCTION: This study aimed to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples by PCR-RFLP. METHODS: Fifty-two strains identified by conventional biochemical exams were submitted to PCR amplification and digested with HinfI. Only 20 (38.5%) of the 52 strains showed a DNA pattern expected for E. gallinarum and E. casseliflavus. RESULTS: Analysis of the results of this study showed that E. gallinarum and E. casseliflavus are occasionally erroneously identified and confirmed the potential application of 16S rDNA analysis for accurate identification of these species. CONCLUSIONS: A correct identification is important to distinguish between intrinsic and acquired vancomycin resistance.

A simple molecular method for rapid identification of commercially used Amblyseius and Neoseiulus species (Acari: Phytoseiidae)

Zootaxa ◽  
2018 ◽  
Vol 4394 (2) ◽  
pp. 270
Author(s):  
M.Y. SYROMYATNIKOV ◽  
A.V. KOKINA ◽  
N.A. BELYAKOVA ◽  
E.G. KOZLOVA ◽  
V.N. POPOV
Keyword(s):  
Molecular Genetic ◽  
Mite Species ◽  
Rapid Identification ◽  
Internal Transcribed Spacers ◽  
Dna Fragments ◽  
Neoseiulus Cucumeris ◽  
Accurate Identification ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Its1 And Its2

Predatory mites from the Amblyseius (Neoseiulus) genus (Family Phytoseiidae, Order Parasitiformes) are widely used for protecting plants against pests, especially thrips. The differentiation of Amblyseius and Neoseiulus species by their morphological features is problematic despite the fact that they are taxonomically different genera. The Phytoseiidae family includes a lot of species that are extremely difficult to distinguish from each other. The discovery of new molecular genetic markers might considerably facilitate the express identification of commercial mite species. Despite their high morphological similarity, the three common commercial Amblyseius and Neoseiulus mite species (Neoseiulus cucumeris, Amblyseius swirskii, and Neoseiulus barkeri) differed significantly in the nucleotide sequences of DNA fragments containing the ITS1 and ITS2 internal transcribed spacers. We found that when PCR products of the amplified DNA fragments from A. swirskii, N. cucumeris, and N. barkeri were treated with a combination of AccB1I, AspLEI, and SspI endonucleases and the resulting products were separated by electrophoresis in agarose gel, the obtained picture was sufficiently specific to provide accurate identification of the analyzed mite species. The lengths of the digestion products were different enough to allow their resolution in agarose gels. The PCR-RFLP method developed by us allows the rapid and accurate identification of commercially used Amblyseius and Neoseiulus species without DNA sequencing. The combination of the three endonucleases (AccB1I, AspLEI, and SspI) and FaeI can be used for the differentiation of all other but less frequently commercially used Phytoseiidae mites. 

scholarly journals Morphological and Molecular Characterization of Toxigenic Aspergillus flavus from Groundnut Kernels in Kenya

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Robert O. Okayo ◽  
Darius O. Andika ◽  
Mathews M. Dida ◽  
George O. K’Otuto ◽  
Bernard M. Gichimu
Keyword(s):  
Molecular Characterization ◽  
Aspergillus Flavus ◽  
Control Strategies ◽  
Its Region ◽  
Morphological Observation ◽  
Its2 Region ◽  
Accurate Identification ◽  
Molecular Approaches ◽  
Microscopic Features ◽  
Toxigenic Strains

Pathogenesis of Aspergillus flavus on important agricultural products is a key concern on human health due to the synthesis and secretion of the hazardous secondary metabolite, aflatoxin. This study identified and further characterized aflatoxigenic A. flavus from groundnuts sampled from sundry shops in Kenya using integrated morphological and molecular approaches. The groundnuts were plated on potato dextrose agar for isolation and morphological observation of A. flavus based on macroscopic and microscopic features. Molecular characterization was done through amplification and comparison of the partial sequence of the ITS1-5.8S-ITS2 region. The expression analysis of aflR, aflS, aflD, aflP, and aflQ genes in the aflatoxin biosynthesis pathways was conducted to confirm the positive identification of A. flavus. The gene expression also aided to delineate toxigenic isolates of A. flavus from atoxigenic ones. Morphologically, 18 isolates suspected to be A. flavus were identified. Out of these, 14 isolates successfully amplified the 500 bp ITS region of A. flavus or Aspergillus oryzae, while 4 isolates were not amplified. All the remaining 14 isolates expressed at least one of the aflatoxigenic genes but only 5 had all the genes expressed. Partial sequencing revealed that isolates 5, 11, 12, 13, and 15 had 99.2%, 97.6%, 98.4%, 97.5%, and 100% homology, respectively, to the A. flavus isolate LUOHE, ITS-5.8S-ITS2, obtained from the NCBI database. The five isolates were accurate identification of atoxigenic A. flavus. Precise identification of toxigenic strains of A. flavus will be useful in establishing control strategies of the fungus in food products.

scholarly journals Authentication of the Herbal Medicine Angelicae Dahuricae Radix Using an ITS Sequence-Based Multiplex SCAR Assay

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2134 ◽  
Author(s):  
Pureum Noh ◽  
Wook Kim ◽  
Sungyu Yang ◽  
Inkyu Park ◽  
Byeong Moon
Keyword(s):  
Herbal Medicines ◽  
Morphological Characteristics ◽  
Pcr Amplification ◽  
Its Region ◽  
Sequencing Analysis ◽  
Accurate Identification ◽  
Republic Of China ◽  
Sequence Characterized Amplified Region ◽  
Angelicae Dahuricae Radix ◽  
Species Specific

The accurate identification of plant species is of great concern for the quality control of herbal medicines. The Korean Pharmacopoeia and the Pharmacopoeia of the People’s Republic of China define Angelicae Dahuricae Radix (Baek-Ji in Korean and Bai-zhi in Chinese) as the dried roots of Angelica dahurica or A. dahurica var. formosana belonging to the family Apiaceae. Discrimination among Angelica species on the basis of morphological characteristics is difficult due to their extremely polymorphic traits and controversial taxonomic history. Furthermore, dried roots processed for medicinal applications are indistinguishable using conventional methods. DNA barcoding is a useful and reliable method for the identification of species. In this study, we sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes in A. dahurica, A. dahurica var. formosana, and the related species A. anomala and A. japonica. Using these sequences, we designed species-specific primers, and developed and optimized a multiplex sequence-characterized amplified region (SCAR) assay that can simply and rapidly identify respective species, and verify the contamination of adulterant depending on the polymerase chain reaction (PCR) amplification without sequencing analysis in a single PCR reaction. This assay successfully identified commercial samples of Angelicae Dahuricae Radix collected from Korean and Chinese herbal markets, and distinguished them from adulterants. This multiplex SCAR assay shows a great potential in reducing the time and cost involved in the identification of genuine Angelicae Dahuricae Radix and adulterant contamination.

scholarly journals An RFLP-Based Technique for Identifying Fungi in the Sooty Blotch and Flyspeck Complex on Apple

Plant Disease ◽  
2008 ◽  
Vol 92 (5) ◽  
pp. 794-799 ◽  
Author(s):  
K. B. Duttweiler ◽  
G. Y. Sun ◽  
J. C. Batzer ◽  
T. C. Harrington ◽  
M. L. Gleason
Keyword(s):  
Agar Plate ◽  
Pcr Amplification ◽  
Rflp Analysis ◽  
Its Region ◽  
Morphological Description ◽  
Sooty Blotch ◽  
Pcr Rflp ◽  
Sooty Blotch And Flyspeck ◽  
Rflp Assay

A restriction fragment length polymorphism (RFLP)-based technique was developed to identify members of the sooty blotch and flyspeck (SBFS) disease complex on apple because these fungi are difficult to identify using agar-plate isolation and morphological description. The method includes polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) using a fungal-specific forward primer (ITS1-F) and an SBFS-specific reverse primer (Myc1-R), followed by digestion of the PCR product by the HaeIII restriction enzyme. When applied to previously identified isolates of 24 SBFS-causing species in nine genera, the PCR-RFLP assay produced 14 unique banding patterns. Different genera never shared the same RFLP pattern. To evaluate performance in vivo, the technique was applied to DNA extracted directly from SBFS colonies on apple fruit from three Iowa orchards. The primers amplified the rDNA of only SBFS fungi, with the exception of a Cladosporium sp.; however, its RFLP banding pattern was distinct from those of SBFS fungi. The majority (60%) of SBFS colonies in the in vivo trial were identified to genus by RFLP analysis. The PCR-RFLP assay greatly streamlined the identification process by minimizing the need for culturing, indicating its value as a tool for field studies of the SBFS complex.

scholarly journals Development of Species/Genus specific Primers for Identification of Three Trichoderma Species and for Detection of Trichoderma Genus

2021 ◽  
Author(s):  
You Zhou ◽  
Jun Wang ◽  
Laying Yang ◽  
Lijia Guo ◽  
Shuting He ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
Elongation Factor ◽  
Pcr Amplification ◽  
Its Region ◽  
Control Agent ◽  
Plant Diseases ◽  
Specific Primers ◽  
Accurate Identification ◽  
Trichoderma Species ◽  
Species Specific

Abstract Trichoderma is a widely used bio-control agent, and has excellent ability to antagonize plant pathogens and promoting plant growth. It has been successfully used to control various plant diseases. The premise of using Trichoderma is accurate identification of it. In this study, four sets of species/genus-specific primers were designed based on the translation elongation factor 1-alpha (tef1) gene and internal transcribed spacer (ITS) region, in order to identify Trichoderma harzianum, T. koningiopsis, and T.virens, and detect the genus of Trichoderma. Here, the rapid, simple, and reliable PCR methods were development using the species-specific primers EHarF2/EHarR2, EKoisF/EKoisR, and EVireF/EVireR to produce 253 bp (tef1 gene), 255 bp (tef1 gene), and 263 bp (tef1 gene) DNA bands to identify T. harzianum, T. koningiopsis, and T. virens, respectively. The genus-specific primers ITricF/ITricR produces a single DNA band in the range of 103–113 bp (ITS region) to distinguish Trichoderma from other genera fungi, and the size of the band is related to the species of Trichoderma. In addition, ITricF/ITricR could use for real-time PCR amplification for detection the quantity of Trichoderma spp..

Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
Author(s):  
Rainer Blasczyk ◽  
Markus Ritter ◽  
Christian Thiede ◽  
Jenny Wehling ◽  
Günter Hintz ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Factor V ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Specific Amplification ◽  
Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

scholarly journals Morpho-molecular identification and first report of Fusarium equiseti in causing chilli wilt from Kashmir (Northern Himalayas)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ammarah Hami ◽  
Rovidha S. Rasool ◽  
Nisar A. Khan ◽  
Sheikh Mansoor ◽  
Mudasir A. Mir ◽  
...  
Keyword(s):  
Dna Barcoding ◽  
Pcr Amplification ◽  
Its Region ◽  
Fungal Species ◽  
Pathogenicity Test ◽  
Kashmir Valley ◽  
Fusarium Equiseti ◽  
Infected Root ◽  
Fusarium Sp ◽  
High Degree

AbstractChilli (Capsicum annuum L.) is one of the most significant vegetable and spice crop. Wilt caused by Fusarium Sp. has emerged as a serious problem in chilli production. Internal transcribed spacer (ITS) region is widely used as a DNA barcoding marker to characterize the diversity and composition of Fusarium communities. ITS regions are heavily used in both molecular methods and ecological studies of fungi, because of its high degree of interspecific variability, conserved primer sites and multiple copy nature in the genome. In the present study we focused on morphological and molecular characterization of pathogen causing chilli wilt. Chilli plants were collected from four districts of Kashmir valley of Himalayan region. Pathogens were isolated from infected root and stem of the plants. Isolated pathogens were subjected to DNA extraction and PCR amplification. The amplified product was sequenced and three different wilt causing fungal isolates were obtained which are reported in the current investigation. In addition to Fusarium oxysporum and Fusarium solani, a new fungal species was found in association with the chilli wilt in Kashmir valley viz., Fusarium equiseti that has never been reported before from this region. The studies were confirmed by pathogenicity test and re-confirmation by DNA barcoding.

scholarly journals PCR-based genotyping assays to detect germline APC variant associated with hereditary gastrointestinal polyposis in Jack Russell terriers

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Kyoko Yoshizaki ◽  
Akihiro Hirata ◽  
Hiroyuki Matsushita ◽  
Naohito Nishii ◽  
Mifumi Kawabe ◽  
...  
Keyword(s):  
Mutant Allele ◽  
False Negative ◽  
Disease Onset ◽  
Pcr Amplification ◽  
Buccal Swab ◽  
Wild Type ◽  
Gastrointestinal Polyposis ◽  
Pcr Rflp ◽  
Formalin Fixed Paraffin Embedded ◽  
Rflp Assay

Abstract Background The prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell terriers (JRTs) has increased in Japan since the late 2000s. Recently, we demonstrated that JRTs with GI polyps harbor identical germline variant in the APC gene (c.[462_463delinsTT]) in the heterozygous state. Thus, this disease is an autosomal dominant hereditary disorder. Although the affected JRTs have distinct features, such as the development of multiple GI polyps and an early age of disease onset, genetic testing is indispensable for a definitive diagnosis. Here, polymerase chain reaction (PCR)-based assays capable of detecting germline APC variant were designed and validated using synthetic wild-type and mutant DNAs and genomic DNAs from carrier and non-carrier dogs. Result First, the PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed by taking advantage of the germline APC variant creating a new restriction site for MseI. In the PCR-RFLP assay, the 156-bp region containing the variant site was amplified by PCR and subsequently digested with MseI, yielding diagnostic 51 and 58 bp fragments from the mutant allele and allowing determination of the APC genotypes. It was possible to determine the genotypes using genomic DNA extracted from the peripheral blood, buccal swab, or formalin-fixed paraffin-embedded tissue. Next, a TaqMan duplex real-time PCR assay was developed, where a 78-bp region flanking the variant was amplified in the presence of wild-type allele- and mutant allele-specific fluorescent probes. Using blood-derived DNA, altogether 40 cycles of PCR amplification determined the APC genotypes of all examined samples by measuring the fluorescence intensities. Importantly, false-positive and false-negative errors were never detected in both assays. Conclusion In this study, we developed highly reliable genetic tests for hereditary GI polyposis in JRTs, providing accurate assessment of the presence of the causative germline APC variant. The genotyping assays could contribute to the diagnosis and prevention of hereditary GI polyposis in dogs.

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