scholarly journals Identification of Monilinia fructigena, M. fructicola, M. laxa, and Monilia polystroma on Inoculated and Naturally Infected Fruit Using Multiplex PCR

Plant Disease ◽  
2004 ◽  
Vol 88 (11) ◽  
pp. 1219-1225 ◽  
Author(s):  
Marie-José Côté ◽  
Marie-Claude Tardif ◽  
Allison J. Meldrum
Keyword(s):  
Multiplex Pcr ◽  
Dna Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Genomic Region ◽  
Monilinia Fructigena ◽  
Identification Method ◽  
Pcr Product ◽  
Pcr Products ◽  
Monilia Polystroma ◽  
Species Specific

Monilinia fructigena, M. fructicola, M. laxa, and Monilia polystroma each have a different regulatory status. To monitor imported and exported fruit for the presence of quarantined Monilinia or Monilia species, a timely identification method is required. Random amplified polymorphic DNA analysis was used to generate an M. fructigena-specific band that was characterized by sequencing. Using the sequence obtained, primers were designed to amplify bands in the same genomic region of M. fructicola and M. laxa. These bands were also characterized by sequencing. From all three sequences, a multiplex polymerase chain reaction (PCR) method based on a common reverse primer (MO368-5) and three species-specific forward primers (MO368-8R, MO368-10R, and Laxa-R2) was established for the differentiation of the three Monilinia species. The multiplex PCR was tested with additional isolates and consistently produced a 402-bp PCR product for M. fructigena, a 535-bp product for M. fructicola, and a 351-bp product for M. laxa. The method was also used with isolates of the recently characterized Monilia polystroma, and all isolates amplified a 425-bp PCR product. The identification method was shown to amplify a PCR product directly from inoculated apples, and the PCR band produced was specific to the inoculated Monilinia or Monilia species. Furthermore, the multiplex PCR was used to identify Monilinia species on naturally infected stone fruits. The method correctly identified infections by both M. laxa and M. fructicola by successful amplification of corresponding PCR products for each species.

scholarly journals Comparison of Two Multiplex PCR Systems for Meat Species Authentication

2019 ◽  
Author(s):  
D. Al-taghlubee ◽  
A. Misaghi ◽  
P. Shayan ◽  
A. Akhondzadeh Basti ◽  
H. Gandomi ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Cross Reactivity ◽  
Specific Pcr ◽  
Pcr Products ◽  
Reverse Primer ◽  
Specific Reverse Primer ◽  
Meat Species ◽  
Polymerase Chain ◽  
Species Authentication ◽  
Species Specific

Background: Meat species adulteration has become a problem of concern. This study aimed to compare two previously published multiplex Polymerase Chain Reaction (PCR) methods for meat species authentication.  Methods: The primers used in the first multiplex PCR involved species-specific reverse primer for sheep, goat, cattle, pig, and donkey with universal forward primer. In the second multiplex PCR, the primers included species-specific forward and reverse primer for pork, lamb, ostrich, horse, and cow. The extracted DNA was then amplified with species-specific primers and with mix primers separately in the respective multiplex PCR. Results: The first multiplex PCR was accompanied with cross reactivity, whereas the second multiplex PCR was specific as expected for pork, lamb, ostrich, horse, and cow. The first set of multiplex PCR showed not always amplification of all species-specific DNAs with a mixture of DNA from mentioned animals. Regarding the second set of primers, the extracted DNA of different meat species was amplified with corresponding species primers as simplex PCR resulting in specific amplicons for species DNA prepared from sheep, ostrich, horse, pig, and cattle with the specific PCR products of 119, 155, 253, 100, and 311 bp, respectively. Conclusion: Based on the present investigation, we recommend the multiplex PCR with the second set of primers included species-specific forward and reverse primers for species authentication of five meat types, including pork, lamb, ostrich, horse, as well as cow.

scholarly journals The use of cytochrome b and ryanodine polymorphism to identify DNA of animal and human origin

2019 ◽  
Author(s):  
Małgorzata Natonek-Wiśniewska ◽  
Anna Radko
Keyword(s):  
Cytochrome B ◽  
Red Deer ◽  
Dna Analysis ◽  
Roe Deer ◽  
Restriction Analysis ◽  
Dna Profile ◽  
Applied Method ◽  
Gene Coding ◽  
Pcr Product ◽  
Pcr Products

The aim of this study was to determine a match between DNA recovered from evidence material, such as knocked down red deer, and from comparative material in form of two brown traces on the bonnet of a car driven by a person suspected of knocking down the animal. The spots coming from the car provided no DNA profile, which questioned that they originated from a red deer and ruled out performance of a comparative DNA analysis. For this reason, the material obtained from the blood smear was analyzed for species identification. The method applied can discriminate between cattle, red deer and roe deer based on restriction analysis (Tsp509I) of PCR product (195bp), obtained by amplifying a fragment of the cytochrome b coding gene. Because the obtained restriction profile confirmed the match with red deer DNA for one trace, and in the second case ruled out that the biological traces originated from the species mentioned above, the PCR products were subjected to sequencing. In both cases, 195bp PCR products that were 98% homologous with red deer DNA sequence-NC_007704.2-trace1 and with the gene coding for the human ryanodine receptor-NC_008799.2-trace2. The quantity and quality of DNA obtained from the traces collected from the car bonnet did not allow confirmation of the involvement of a specific animal in the event, but the applied method made it possible to determine the species from which the obtained traces originated. Furthermore, the applied method, which was used earlier to determine cervine DNA, was successfully used to detect human DNA.

scholarly journals A Multiplex PCR System for the Specific Detection of Cylindrocarpon liriodendri, C. macrodidymum, and C. pauciseptatum from Grapevine

Plant Disease ◽  
2009 ◽  
Vol 93 (8) ◽  
pp. 821-825 ◽  
Author(s):  
Sandra Alaniz ◽  
Josep Armengol ◽  
José García-Jiménez ◽  
Paloma Abad-Campos ◽  
Maela León
Keyword(s):  
Multiplex Pcr ◽  
Extraction Methods ◽  
Specific Pcr ◽  
Pcr Product ◽  
Total Dna ◽  
Polymerase Chain ◽  
Foot Disease ◽  
Species Specific ◽  
Black Foot ◽  
Black Foot Disease

Cylindrocarpon liriodendri and C. macrodidymum are the causal agents of grapevine black foot disease. Recently, a third species, C. pauciseptatum, has been isolated from roots of grapevine showing decline symptoms. Currently, reliable identification of isolates of these species through phenotypical characteristics has not been possible. The polymerase chain reaction (PCR)-based method developed in this study allows a quick and easy detection of Cylindrocarpon spp. associated with grapevine. Three primer pairs annealing to variable, partly species-specific sites of the internal transcribed spacer regions amplified species-specific PCR fragments of different sizes in C. liriodendri, C. macrodidymum, and C. pauciseptatum in a multiplex assay with DNA obtained with both quick and traditional extraction methods. They did not generate any PCR product in other fungal trunk pathogens or contaminants commonly associated with grapevines. When universal fungal ITS primers were used in a nested multiplex PCR, the three primer pairs also detected C. liriodendri, C. macrodidymum, and C. pauciseptatum in total DNA extracted from roots of inoculated grapevines. The designed methods can be used for the diagnosis of these fungi from pure culture or infected grapevines.

scholarly journals Suitability of different primers for specific molecular detection of Monilinia spp.

2017 ◽  
Vol 62 (2) ◽  
pp. 167-177
Author(s):  
Natasa Duduk ◽  
Miljan Vasic ◽  
Nina Vuckovic ◽  
Aleksandra Zebeljan ◽  
Ivana Vico
Keyword(s):  
Molecular Detection ◽  
Apple Fruit ◽  
Stone Fruits ◽  
Specific Detection ◽  
Specific Primers ◽  
Monilinia Fructigena ◽  
Detection And Identification ◽  
Reverse Primer ◽  
Monilia Polystroma ◽  
Species Specific

Monilinia spp. are economically important pathogens of pome and stone fruits. Four Monilinia species are present in Serbia - Monilinia fructigena, M. laxa, M. fructicola and Monilia polystroma. As detection and identification of Monilinia species are complex, the aim of this research was to evaluate species-specific primers in PCR in order to standardize fast and reliable molecular methods for differentiation between the four Monilinia species. Isolates of M. fructigena, M. laxa, M. fructicola and M. polystroma from apple fruit and referent isolates from Italy and Japan were used for testing. Specific molecular detection of M. laxa was obtained using ITS1Mlx/ITS4Mlx and Ml-Mfg-F2/Ml-Mfc-R1 primer pairs, and M. fructicola using ITS1Mfcl/ITS4Mfcl and Mfc-F1/Mfc-R1 primer pairs. ITS1Mfgn/ITS4Mfgn and ITS1/Mfg-R2 primer pairs, described as M. fructigena species-specific, amplified M. fructigena and M. polystroma, as well. Specific detection of these two species as well as of all four tested Monilinia species was obtained using the reverse primer MO368-5 with forward primers MO368-8R, Laxa-R2 and MO368-10R in separate or in Multiplex PCR reactions.

scholarly journals Identification and differentiation among chicken’s, duck’s, quail’s, rabbit’s and turkey's meat using PCR-RFLP technique

2015 ◽  
Vol 31 (1) ◽  
pp. 101-108 ◽  
Author(s):  
S.M. Abdel-Rahman ◽  
A.M. Elmaghraby ◽  
A.S. Haggag
Keyword(s):  
Mitochondrial Dna ◽  
Cytochrome B ◽  
Fragment Length ◽  
Restriction Analysis ◽  
Restriction Enzymes ◽  
Pcr Product ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Species Specific ◽  
Rflp Assay

PCR-RFLP technique was developed for identification and differentiation among chicken?s, duck?s, quail?s, rabbit?s and turkey's meat. DNA from small amount of muscles (0.05 g) was extracted and a region of mitochondrial DNA (cytochrome-b gene) in chicken, duck, quail, rabbit and turkey was amplified by PCR. Fragment length of the PCR product was 371 bp in chicken, 374 bp in duck and rabbit and 377 bp in both quail and turkey. Six nucleotides different makes it difficult to differentiate among these five species-specific meat. For differentiation, three different restriction enzymes (DdeI, MspI and TaqI) were used to digest the PCR products. Restriction analysis showed difference among chicken?s, duck?s, quail?s, rabbit?s and turkey's meat. Where, DdeI yielded two fragments (291 and 83 bp) only in rabbit?s meat. MspI yielded three fragments (221, 85 and 65 bp) in chicken?s meat and two fragments (290 and 87 bp) in both quail?s and turkey's meat. TaqI yielded three fragments (146, 134 and 94 bp) in duck?s meat and two fragments (226 and 151 bp) in quail?s meat. The use of Cytb- PCR-RFLP assay allowed a direct and fast authentication and differentiation among chicken?s, duck?s, quail?s, rabbit?s and turkey's meat.

scholarly journals Development of a Multiplex PCR-Based Assay for Rapid Serotyping of Erysipelothrix Species

2020 ◽  
Vol 58 (6) ◽  
Author(s):  
Yoshihiro Shimoji ◽  
Kazumasa Shiraiwa ◽  
Haruka Tominaga ◽  
Sayaka Nishikawa ◽  
Masahiro Eguchi ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Epidemiological Studies ◽  
Genomic Region ◽  
Erysipelothrix Rhusiopathiae ◽  
Content Type ◽  
Heat Stable ◽  
Wide Range ◽  
Species Specific ◽  
Specific Rabbit

ABSTRACT The Gram-positive bacterium Erysipelothrix rhusiopathiae is a zoonotic pathogen that causes erysipelas in a wide range of mammalian and avian species. Historically, E. rhusiopathiae has been differentiated from other Erysipelothrix species by serotyping. Among 28 serovars of Erysipelothrix species, specific serovars, namely, 1a, 1b, and 2 of E. rhusiopathiae, are associated mainly with the disease in pigs, poultry, and humans; however, other serovar strains are often simultaneously isolated from diseased and healthy animals, indicating the importance of isolate serotyping for epidemiology. The traditional serotyping protocol, which uses heat-stable peptidoglycan antigens and type-specific rabbit antisera in an agar-gel precipitation test, is time-consuming and labor-intensive. To develop a rapid serotyping scheme, we analyzed sequences of the 12- to 22-kb chromosomal region, which corresponds to the genetic region responsible for virulence of serovar 1a and 2 strains of E. rhusiopathiae, of the 28 serovars of Erysipelothrix species. We confirmed that the serovar 13 strain lacks the genomic region and that some serovar strains possess very similar or the same genetic structure, prohibiting differentiation of the serovars. We created 4 multiplex PCR sets allowing the simultaneous detection and differentiation of the majority of Erysipelothrix serovars. Together with a previously reported multiplex PCR that can differentiate serovars 1a, 1b, 2, and 5, the multiplex PCR-based assay developed in this study covers all but one (serovar 13) of the reported serovars of Erysipelothrix species and should be a valuable tool for etiological as well as epidemiological studies of Erysipelothrix infections.

scholarly journals (271) Expression of the NRT1 Gene in Cornus and Rhododendron

HortScience ◽  
2005 ◽  
Vol 40 (4) ◽  
pp. 1020C-1020
Author(s):  
David R. Sandrock ◽  
Anita N. Azarenko ◽  
Ruth M. Martin ◽  
Nahla V. Bassil
Keyword(s):  
Nitrate Uptake ◽  
Transport Proteins ◽  
Rt Pcr ◽  
Degenerate Primers ◽  
Specific Primers ◽  
Nucleotide Level ◽  
Pcr Product ◽  
Pcr Products ◽  
Species Specific ◽  
Hydroponically Grown

The NRT1gene family encodes transport proteins with dual or low affinity for nitrate. The objectives of this experiment were to develop a system that could be used to compare the expression of the NRT1genes between species. This was accomplished by comparing sequences of NRT1homologues from various species and designing degenerate primers in regions of high homology. These primers were used to amplify a region of the NRT1gene from species of interest. A 635 bp PCR product was amplified from each species using the MD2-1 (5' ATGTTACCAAYWTGGGCMAC-3') and MD2-2 (5'-GCCAMWARCCARTAGAAAT-3') primers. The PCR products were cloned and sequenced. At the nucleotide level, CornussericeaL. `Kelseyi' and RhododendronL. `Unique' were 79.52% identical. Species-specific primers were designed and used for RT-PCR to compare NRT1expression in roots of hydroponically grown C. sericea, C. sericea `Kelseyi', and Rhododendron`Unique'. The relative levels of NRT1expression, normalized using 18S rRNA as a standard, were ≈3.2 to 1.7 to 1.0 for C. sericea, C. sericea `Kelseyi', and Rhododendron`Unique', respectively. This approach may eventually be used to examine nitrate uptake potential in different taxa of plants at different times during the growing season.

Abnormal Processing of the Glycoprotein IIb Transcript due to a Nonsense Mutation in Exon 17 Associated with Glanzmann’s Thrombasthenia

1995 ◽  
Vol 73 (05) ◽  
pp. 756-762 ◽  
Author(s):  
Yoshiaki Tomiyama ◽  
Hirokazu Kashiwagi ◽  
Satoru Kosugi ◽  
Masamichi Shiraga ◽  
Yoshio Kanayama ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Molecular Genetic ◽  
Genetic Defect ◽  
Nucleotide Sequence Analysis ◽  
Type I ◽  
Normal Amount ◽  
Immunoblot Assay ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia ◽  
Pcr Products

SummaryWe analyzed the molecular genetic defect responsible for type I Glanzmann’s thrombasthenia in a Japanese patient. In an immunoblot assay using polyclonal anti-GPIIb-IIIa antibodies, some GPIIIa (15% of normal amount) could be detected in the patient’s platelets, whereas GPIIb could not (<2% of normal amount). Nucleotide sequence analysis of platelet GPIIb mRNA-derived polymerase chain reaction (PCR) products revealed that patient’s GPIIb cDNA had a 75-bp deletion in the 3’ boundary of exon 17 resulting in an in-frame deletion of 25 amino acids. DNA analysis and family study revealed that the patient was a compound heterozygote of two GPIIb gene defects. One allele derived from her father was not expressed in platelets, and the other allele derived from her mother had a 9644C → T mutation which was located at the position -3 of the splice donor junction of exon 17 and resulted in a termination codon (TGA). Moreover, quantitative analysis demonstrated that the amount of the abnormal GPIIb transcript in the patient’s platelets was markedly reduced. Thus, the C → T mutation resulting in the abnormal splicing of GPIIb transcript and the reduction in its amount is responsible for Glanzmann’s thrombasthenia.

EVALUATION OF PTYCHOBOTHRIDEAN CESTODES USING RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) MARKERS FROM FRESH WATER FISHES IN GODAVARI BASIN (M.S.) INDIA

2017 ◽  
Vol 23 (2) ◽  
Author(s):  
SUNITA BORDE ◽  
ASAWARI FARTADE ◽  
AMOL THOSAR ◽  
RAHUL KHAWAL
Keyword(s):  
Fresh Water ◽  
Rapd Markers ◽  
Random Amplified Polymorphic Dna ◽  
Band Pattern ◽  
Genomic Variation ◽  
Polymorphic Band ◽  
Rapd Profile ◽  
Band Size ◽  
Pcr Product ◽  
The Common

Ptychobothridean genera like Senga and Circumoncobothrium are the common parasites of fresh water fishes. The genotypic study of these parasites was taken by RAPD. The RAPD profile of these two parasites were not similar to each other as depicted by the band pattern in picture. These results suggest the presence of inter-specific polymorphism among cestode parasites of two different genera for RAPD analysis. The present study demonstrated that genetic differentiation of cestode parasites could be accomplished on the basis of genomic variation with polymorphic band pattern using RAPD. All the detected bands (PCR product) were polymorphic and band size ranged from 500-5000 bp in length. The RAPD of profiles using GBO-31, GBO-32, GBO-33, GBO-34, GBO-35 and GBO-36. Primers were able to characterize inter-specific polymorphism among the two genus ( Senga and Circumoncobothrium ). Genetic analysis suggests that Senga and Circumoncobothrium show genetic diversity with respect to RAPD patterns using all the six primers used for the present study. The genetic distance between the analyzed genuses ranged from 0.14 to 0.80. The differentiation of the two parasites on the basis of genetic markers could greatly facilitate study on the biology of these parasites.

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