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 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 Detection of Phomopsis sclerotioides in Commercial Cucurbit Field Soil by Nested Time-Release PCR

Plant Disease ◽  
2012 ◽  
Vol 96 (4) ◽  
pp. 515-521 ◽  
Author(s):  
T. Ito ◽  
S. Fuji ◽  
E. Sato ◽  
Y. Iwadate ◽  
T. Toda ◽  
...  
Keyword(s):  
Minimum Concentration ◽  
Disease Symptoms ◽  
Specific Pcr ◽  
Phomopsis Sclerotioides ◽  
Fluorescent Pcr ◽  
Pcr Products ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Species Specific ◽  
Pcr Techniques

A polymerase chain reaction (PCR)-based molecular method to detect Phomopsis sclerotioides in soil was developed using a species-specific primer pair. To improve sensitivity of the detection, three PCR techniques were used; namely, nested PCR using the primer pair internal transcribed spacer (ITS)1 and ITS4, time-release PCR using two different DNA polymerases (recombinant Taq and AmpliTaq Gold), and fluorescent PCR to obtain fluorescent-labeled PCR products that can be analyzed by capillary electrophoresis. The latter two techniques were combined and termed nested time-release fluorescent (NTRF)-PCR. The minimum concentration of DNA required to obtain species-specific PCR products successfully was 50 fg/μg. Using the NTRF-PCR method, the fungus could be detected in sandy soil that was artificially infested at a density of 10 CFU/g. The pathogen was detected in most soil samples collected from commercial cucumber fields in which visual disease symptoms had appeared, and even in samples collected from fields where visual disease symptoms had not appeared. To prevent the invasion and establishment of root-inhabiting pathogens such as P. sclerotioides, it is critical to detect the fungus in soil as soon as possible after its introduction into a cucumber-growing region.

scholarly journals A Comparative Study of 5S rDNA Non-Transcribed Spacers in Elaeagnaceae Species

Plants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 4
Author(s):  
Oleg S. Alexandrov ◽  
Olga V. Razumova ◽  
Gennady I. Karlov
Keyword(s):  
Polymerase Chain Reaction ◽  
Molecular Markers ◽  
Dna Markers ◽  
5S Rdna ◽  
Chain Reaction ◽  
Closely Related Species ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Species Specific ◽  
Shepherdia Canadensis

5S rDNA is organized as a cluster of tandemly repeated monomers that consist of the conservative 120 bp coding part and non-transcribed spacers (NTSs) with different lengths and sequences among different species. The polymorphism in the 5S rDNA NTSs of closely related species is interesting for phylogenetic and evolutional investigations, as well as for the development of molecular markers. In this study, the 5S rDNA NTSs were amplified with universal 5S1/5S2 primers in some species of the Elaeagnaceae Adans. family. The polymerase chain reaction (PCR) products of five Elaeagnus species had similar lengths near 310 bp and were different from Shepherdia canadensis (L.) Nutt. and Sh. argentea (Pusch.) Nutt. samples (260 bp and 215 bp, respectively). The PCR products were cloned and sequenced. An analysis of the sequences revealed that intraspecific levels of NTS identity are high (approximately 95–96%) and similar in the Elaeagnus L. species. In Sh. argentea, this level was slightly lower due to the differences in the poly-T region. Moreover, the intergeneric and intervarietal NTS identity levels were studied and compared. Significant differences between species (except E. multiflora Thunb. and E. umbellata Thunb.) and genera were found. Herein, a range of the NTS features is discussed. This study is another step in the investigation of the molecular evolution of Elaeagnaceae and may be useful for the development of species-specific DNA markers in this family.

scholarly journals A 16S rDNA-based nested PCR protocol to detect Campylobacter gracilis in oral infections

2003 ◽  
Vol 17 (2) ◽  
pp. 142-146 ◽  
Author(s):  
José Freitas Siqueira Júnior ◽  
Isabela das Neves Rôças
Keyword(s):  
16S Rdna ◽  
High Sensitivity ◽  
High Specificity ◽  
Cross Reactivity ◽  
Nested Polymerase Chain Reaction ◽  
Oral Infections ◽  
Root Canals ◽  
Infected Root ◽  
Pcr Products ◽  
Species Specific

The aim of this study was to describe a 16S rDNA-based nested polymerase chain reaction (nPCR) assay to investigate the occurrence of Campylobacter gracilis in oral infections. Samples were collected from ten infected root canals, ten cases of acute periradicular abscesses and eight cases of adult marginal periodontitis. DNA extracted from the samples was initially amplified using universal 16S rDNA primers. A second round of amplification used the first PCR products to detect C. gracilis using oligonucleotide primers designed from species-specific 16S rDNA signature sequences. The nPCR assay used in this study showed a detection limit of 10 C. gracilis cells and no cross-reactivity was observed with nontarget bacteria. C. gracilis was detected in the three types of oral infections investigated - 4/10 infected root canals; 2/10 acute periradicular abscesses; and 1/8 subgingival specimens from adult periodontitis. The method proposed in this study showed both high sensitivity and high specificity to directly detect C. gracilis in samples from root canal infections, abscesses, and subgingival plaque. Our findings confirmed that C. gracilis may be a member of the microbiota associated with distinct oral infections, and its specific role in such diseases requires further clarification.

scholarly journals Characterization of a New Alloantigen (SH) on the Human Neutrophil Fcγreceptor IIIb

Blood ◽  
1997 ◽  
Vol 89 (3) ◽  
pp. 1027-1034 ◽  
Author(s):  
Juergen Bux ◽  
Ernst-Ludwig Stein ◽  
Philippe Bierling ◽  
Patricia Fromont ◽  
Mary Clay ◽  
...  
Keyword(s):  
Nucleotide Sequence Analysis ◽  
Polymorphism Analysis ◽  
Mutational Event ◽  
Coding Region ◽  
Specific Pcr ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Antigen Frequency ◽  
Neonatal Neutropenia

Abstract Polymorphic structures of the neutrophil Fcγreceptor IIIb (FcγRIIIb) result in alloantibody formation that causes alloimmune neonatal neutropenia and transfusion reactions. Alloantigens located on FcγRIIIb include the antigens NA1 and NA2. In four cases of alloimmune neonatal neutropenia, granulocyte-specific alloantibodies directed against a thus far unknown antigen were detected by granulocyte agglutination and immunofluorescence tests in the maternal sera. By the use of the monoclonal antibody–specific immobilization of granulocyte antigens (MAIGA) assay, the new antigen, termed SH, was located on the FcγRIIIb. Nucleotide sequence analysis of the FcγRIIIb coding region from a SH(+) individual showed a single-base C→A mutation at position 266, which results in an Ala78Asp amino acid substitution. A family study confirmed that this nucleotide difference is inherited, and corresponds to the SH phenotype. Serologic typing of 309 randomly selected individuals showed an antigen frequency of 5% in the white population. The same frequency was found by genotyping, for which a technique based on polymerase chain reaction (PCR) using sequence-specific primers (PCR-SSP) was developed. Typing of all SH(+) individuals for NA1 and NA2, and PCR-restriction fragment length polymorphism analysis of the NA-specific PCR products from five SH(+) individuals using the SH-specific endonuclease SfaN I showed that SH antigen is very probably the result of an additional mutational event in the NA2 form of the FcγRIIIB gene. Immunochemical studies also demonstrated that the SH determinants reside on the 65- to 80-kD NA2 isoform of the FcγRIIIb. Our findings show the existence of an additional polymorphism of the FcγRIIIb, which can result in alloantibody formation causing alloimmune neonatal neutropenia.

Microsporidial infections due toEncephalitozoon intestinalisin non-HIV-infected patients with chronic diarrhoea

2011 ◽  
Vol 140 (10) ◽  
pp. 1773-1779 ◽  
Author(s):  
J. YAKOOB ◽  
Z. ABBAS ◽  
M. ASIM BEG ◽  
W. JAFRI ◽  
S. NAZ ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Stool Examination ◽  
Chronic Diarrhoea ◽  
Healthy Controls ◽  
Chain Reaction ◽  
Specific Primers ◽  
Specific Pcr ◽  
Stool Samples ◽  
Polymerase Chain ◽  
Species Specific

SUMMARYWe determined the prevalence of microsporidiaEnterocytozoon(Ent.)bieneusiandEncephalitozoon(E.)intestinalisinfection in patients with chronic diarrhoea and hepatocellular carcinoma (HCC). A total of 330 stool samples were examined from 171 (52%) patients with chronic diarrhoea, 18 (5%) with HCC while 141 (43%) were controls. Stool microscopy, polymerase chain reaction (PCR) with species-specific primers forEnt. bieneusiandE. intestinalisand sequencing were carried out. Microsporidia were found by trichrome staining in 11/330 (3%) andE. intestinalisby PCR in 13/330 (4%) whileEnt. bieneusiwas not detected. PCR forE. intestinaliswas positive in 8/171 (5%) stool samples from patients with chronic diarrhoea, 2/141 (1·4%) samples from healthy controls and in 3/18 (17%) samples from patients with HCC. In the chronic diarrhoea group,E. intestinaliswas positive in 4/171 (2·3%) (P=0·69) stool samples compared to 2/18 (11%) (P=0·06) in the HCC group and 2/141 (1·4%) from healthy controls.E. intestinalisinfection was significantly associated with chronic diarrhoea and HCC in these patients who were negative for HIV. Stool examination with trichrome or species-specific PCR for microsporidia may help establish the cause of chronic diarrhoea.

A simple method for discriminating Bursaphelenchus xylophilus and B. mucronatus by species-specific polymerase chain reaction primer pairs

Nematology ◽  
2004 ◽  
Vol 6 (2) ◽  
pp. 273-277 ◽  
Author(s):  
Koji Matsunaga ◽  
Katsumi Togashi
Keyword(s):  
Pcr Amplification ◽  
Nematode Species ◽  
Bursaphelenchus Xylophilus ◽  
Polymerase Chain Reaction Primer ◽  
Dna Fragments ◽  
Simple Method ◽  
Specific Pcr ◽  
Pcr Products ◽  
Specific Pcr Primer ◽  
Species Specific

Abstract Two species-specific PCR primer pairs were developed for identifying the two nematode species, Bursaphelenchus xylophilus and B. mucronatus. The primer pairs were developed from the sequence of ribosomal DNA (rDNA) repeats to produce DNA fragments of different lengths by PCR amplification. The DNA fragments for B. mucronatus and B. xylophilus were 210 bp and 557 bp, respectively. When mixed, neither primer pair inhibited the PCR amplification of the other. Five isolates of B. xylophilus and four isolates of B. mucronatus showed different band profiles of PCR products between the two species, but identical profiles among isolates of the same species.

Estimating the prevalence of mixed-type gonococcal infections in Queensland, Australia

Sexual Health ◽  
2015 ◽  
Vol 12 (5) ◽  
pp. 439 ◽  
Author(s):  
Ella Trembizki ◽  
Christine Doyle ◽  
Cameron Buckley ◽  
Amy Jennison ◽  
Helen Smith ◽  
...  
Keyword(s):  
Sequence Data ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Anatomical Site ◽  
Diagnostic Screening ◽  
Specific Pcr ◽  
Screening Population ◽  
Pcr Products ◽  
Polymerase Chain ◽  
The One

Background Mixed gonococcal infections within the one anatomical site have been recognised but questions remain over how often they occur. In this study, the aim was to estimate the prevalence of mixed gonococcal infections using novel real-time polymerase chain reaction (PCR) methods that were developed and validated, targeting the gonococcal porB gene. Methods: Neisseria gonorrhoeae strains were categorised into three different porB groups, based on sequence data derived from N. gonorrhoeae multi-antigen sequence typing (NG-MAST) analyses of local isolates. Specific PCR methods for each group were then developed and these PCR methods were used to test clinical samples (n = 350) that were positive for gonorrhoea as determined by nucleic acid amplification test (NAAT) diagnostic screening. Results: Initial validation using isolates showed the group PCR methods proved 100% sensitive and 100% specific for their respective porB groups. When applied to the clinical specimens, 298/350 (85%) provided positive results by the group PCR methods. Of these, four specimens showed evidence of mixed infections, supported by subsequent DNA sequencing of the PCR products. Conclusions: The data provide further evidence of mixed gonococcal infections at the same anatomical site, but show that such infections may be relatively infrequent (1.3%; 95% confidence interval 0.01–2.6%) in a general screening population.

Identification of starter cultures in thermally treated plain yogurt using gene probes and polymerase chain reaction

1996 ◽  
Vol 63 (4) ◽  
pp. 607-613 ◽  
Author(s):  
Sonja Lick ◽  
Martina Keller ◽  
Uli Krusch ◽  
Knut J. Heller
Keyword(s):  
Polymerase Chain Reaction ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Lactobacillus Delbrueckii ◽  
Agarose Gel Electrophoresis ◽  
Chain Reaction ◽  
Specific Pcr ◽  
Specific Hybridization ◽  
Polymerase Chain ◽  
Species Specific

SummaryPlain yogurts (set type) fermented by three different starter cultures were subjected to different heat treatments (20 s at 72, 80 or 100 ·C, or 30 min at 60, 70, 80 or 100 ·C). DNA was extracted from each yogurt and analysed by agarose gel electrophoresis, by species-specific hybridization and by primer-specific polymerase chain reaction (PCR). Obvious degradation of DNA could be observed after heating at 80 ·C for 30 min and at 100 ·C for 20 s and 30 min. Identification ofLactobacillus delbrueckiiusing a species-specific hybridization fragment could be carried out in yogurt treated at 100 ·C for 20 s or for 30 min at lower temperatures. After treatment for 30 min at 100 ·C identification by hybridization was no longer possible. However, under the same conditions identification of starter microorganisms was still possible by primer-specific PCR, and this was demonstrated as an example forStreptococcus thermophilus.

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