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 Real-Time PCR for Detection and Identification of Anguina funesta, A. agrostis, A. tritici, and A. pacificae

Plant Disease ◽  
2015 ◽  
Vol 99 (11) ◽  
pp. 1584-1589 ◽  
Author(s):  
Wenbin Li ◽  
Zonghe Yan ◽  
Mark K. Nakhla ◽  
Andrea M. Skantar
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
The United States ◽  
Grass Species ◽  
Specific Primers ◽  
Accurate Identification ◽  
Detection And Identification ◽  
Stem Gall ◽  
Species Specific

A number of seed, leaf, and stem gall nematodes are of significance to the forage and landscape grass and livestock industries. In North America, the bentgrass nematode, Anguina agrostis, reduces seed production on Agrostis tenuis and several other grass species. Anguina funesta is a seed-gall nematode that is most significant for its association with the toxigenic bacteria Rathayibacter toxicus. The wheat seed gall nematode A. tritici causes significant damage to wheat and other cereals; although it has been found in many countries worldwide, it has not been detected in the United States since 1975. Molecular methods based upon sequence variation in the ribosomal internal spacer region are useful for accurate identification of Anguina spp. Described herein are new species-specific primers and TaqMan probes for real-time polymerase chain reaction (PCR) identification of A. agrostis, A. funesta, A. tritici, and A. pacificae. Primer and probe combinations were each specific for the intended species and were sensitive enough to detect as few as 1.25 copies of nematode ribosomal DNA. PCR was also specific and sensitive in duplex assays that included genus-specific internal control primers as well as species-specific primers and probes. These standardized real-time PCR protocols should facilitate fast and accurate identification of Anguina spp. by diagnostic laboratories.

scholarly journals Species-Specific Detection and Identification of Fusarium Species Complex, the Causal Agent of Sugarcane Pokkah Boeng in China

PLoS ONE ◽  
2014 ◽  
Vol 9 (8) ◽  
pp. e104195 ◽  
Author(s):  
Zhenyue Lin ◽  
Shiqiang Xu ◽  
Youxiong Que ◽  
Jihua Wang ◽  
Jack C. Comstock ◽  
...  
Keyword(s):  
Species Complex ◽  
Fusarium Species ◽  
Causal Agent ◽  
Specific Detection ◽  
Pokkah Boeng ◽  
Detection And Identification ◽  
Species Specific

scholarly journals Direct Detection and Identification of the Most Common Bacteria and Fungi Causing Otitis Externa by a Stepwise Multiplex PCR

2021 ◽  
Vol 11 ◽  
Author(s):  
Shima Aboutalebian ◽  
Kazem Ahmadikia ◽  
Hamed Fakhim ◽  
Javaher Chabavizadeh ◽  
Ahmadreza Okhovat ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Otitis Externa ◽  
Fungal Pathogens ◽  
Direct Detection ◽  
Specific Primers ◽  
Multiplex Pcr Assay ◽  
Detection And Identification ◽  
Amplicon Size ◽  
Bacteria And Fungi ◽  
Species Specific

BackgroundConsidering the importance of differential diagnosis of infectious otitis externa (OE), a stepwise PCR-based assay using universal and genus- or species-specific primers for the detection/identification of the most prevalent bacterial and fungal OE was developed and evaluated on the ear aspiration specimens of clinically suspected patients.Methods and MaterialsA total of 120 ear aspiration specimens with otomycosis suspicion were subjected to manual DNA extraction using phenol–chloroform extraction after tissue digestion with a lysis buffer. The multiplex PCR was initially performed using pan-fungal and bacterial homemade primers. Pseudomonas and Staphylococcus specific primers were simultaneously used in one reaction mixture to identify the bacterial genera. Furthermore, for the identification of fungal agents, Candida species-specific multiplex primers targeting the most clinically important Candida species causing OE (i.e., C. albicans, C. parapsilosis, and C. auris), as well as Aspergillus related multiplex PCR identifying the most prevalent Aspergillus species were used in two separate reaction mixtures. All the results of multiplex PCR were interpreted based on the amplicon size.ResultsThe overall multiplex PCR-based detection rate of bacterial (n = 88; 73.3%) and fungal (n = 97; 81%) OE was documented to be 100% along with and complete consistency with the results of direct examination and Giemsa staining. Double amplicon bands of bacterial and fungal pathogens were evidenced in 76 specimens (63.3%). Moreover, the positivity rate of pan-fungal PCR was higher than that of the culture result. Out of 88 pan-bacterial positive PCR specimens, 66 and 47 ones were positive for Staphylococcus and Pseudomonas, respectively. In addition, 30 samples exhibited mixed infection of both, and five specimens remained negative. Out of 97 pan-fungal positive PCR specimens, 67 and 51 ones contained Candida and Aspergillus species, respectively. It should be noted that dual amplicon bands of Candida and Aspergillus-related multiplex PCR were yielded in 30 specimens.ConclusionThe stepwise multiplex PCR assay proved to be more sensitive, more rapid, as well as less cumbersome in detection and identification of fungal and bacterial OE, compared to culture.

scholarly journals Development of PCR-Based Detection System for Soft Rot Pectobacteriaceae Pathogens Using Molecular Signatures

2020 ◽  
Vol 8 (3) ◽  
pp. 358
Author(s):  
Md Niamul Kabir ◽  
Ali Taheri ◽  
C. Korsi Dumenyo
Keyword(s):  
Pathogen Detection ◽  
Detection System ◽  
Soft Rot ◽  
Pcr Analysis ◽  
Morphological Identification ◽  
Specific Primers ◽  
Detection And Identification ◽  
Significant Yield ◽  
Rot Disease ◽  
Species Specific

Pectobacterium and Dickeya species, usually referred to as soft rot Enterobacteriaceae, are phytopathogenic genera of bacteria that cause soft rot and blackleg diseases and are responsible for significant yield losses in many crops across the globe. Diagnosis of soft rot disease is difficult through visual disease symptoms. Pathogen detection and identification methods based on cultural and morphological identification are time-consuming and not always reliable. A polymerase chain reaction (PCR)-based detection method with the species-specific primers is fast and reliable for detecting soft rot pathogens. We have developed a specific and sensitive detection system for some species of soft rot Pectobacteriaceae pathogens in the Pectobacterium and Dickeya genera based on the use of species-specific primers to amplify unique genomic segments. The specificities of primers were verified by PCR analysis of genomic DNA from 14 strains of Pectobacterium, 8 strains of Dickeya, and 6 strains of non-soft rot bacteria. This PCR assay provides a quick, simple, powerful, and reliable method for detection of soft rot bacteria.

Molecular Detection and Epidemiology of Equine Herpesvirus 2 and 5 in Working Equids in Central Ethiopia

2021 ◽  
Author(s):  
Kifle Wondimagegnehu ◽  
Samson Leta ◽  
Kebede Amenu ◽  
Haileleul Negussie
Keyword(s):  
Respiratory Disease ◽  
Molecular Detection ◽  
Clinical Signs ◽  
Pcr Amplification ◽  
Respiratory Illness ◽  
Working Capacity ◽  
Specific Primers ◽  
Central Ethiopia ◽  
Species Specific ◽  
Working Equids

Abstract Background: Equine respiratory illness is a common problem that impacts the performance of the working capacity of equids. In Ethiopia, respiratory disease is the most common presenting complaint at veterinary clinics and a priority concern for equid owners and veterinary practitioners. Although studies had been conducted in EHV-2 and EHV-5 elsewhere in the world, many unknowns remained. Thus, a detailed study is needed to understand more about the epidemiology of the viruses. This study aimed to detect EHV-2 and EHV-5 from working equids in central Ethiopia. Methods: Nasopharyngeal swabs were collected from 58 horses and donkeys to detect EHV-2 and EHV-5 using PCR. Viral DNA was extracted and PCR amplification was performed using virus-specific primers targeting the conserved region of glycoprotein B (gB) genes.Results: From 58 equids, EHV-5 and EHV-2 were detected in 20 (34.5%) and 19 (32.8%) equids, respectively. Concurrent infection with EHV-2 and EHV-5 was found in 6 (10.3%) diseased equids. EHV-2 was detected in a significantly higher proportion (P = 0.002) in horses (54.5%; n = 18) than donkeys (4%; n = 1). In contrast, a significantly higher (P = 0.004) proportion of EHV-5 was detected in donkeys (56%; n =14) than horses (18.2%; n = 6). EHV-2 was detected in a significantly higher (P = 0.006) proportion in equids displaying signs of respiratory disease (16/33; 48.5%) compared to those without disease (3/25; 12%). EHV-2 positive equids were seven times more likely to display clinical signs of respiratory disease than EHV-2-negative equids (OR = 6.9; 95% CI: 1.72-27.60). For EHV-5, the observed difference was not statistically significant (P = 0.832). A significantly higher (P = 0.041) proportion of EHV-2 was detected in equids living in midland (52.9%; n = 9) compared with highland (24.4%; n = 10). Equids residing in the midland were four times more likely to be exposed to EHV-2 than highland equids (OR = 3.97; 95% CI: 1.05 - 14.89). Conclusion: EHV-2 and EHV-5 are highly prevalent both in horses and donkeys residing in central Ethiopia. Species-specific susceptibility differences in EHV-2 and EHV-5 infection are observed. The observed causal association between EHV-2-test-positive and the appearance of clinical signs of respiratory disorders should be further investigated.

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.

Cloth-Based Hybridization Array System for the Detection and Identification of Ruminant Species in Animal Feed

2006 ◽  
Vol 69 (2) ◽  
pp. 453-458 ◽  
Author(s):  
JENNIFER ARMOUR ◽  
BURTON W. BLAIS
Keyword(s):  
Dna Sequences ◽  
Animal Feed ◽  
Chromogenic Substrate ◽  
Specific Detection ◽  
Substrate Solution ◽  
Ruminant Species ◽  
Meat Meal ◽  
Detection And Identification ◽  
Species Specific ◽  
Peroxidase Conjugate

A cloth-based hybridization array system for the detection and identification of material derived from several ruminant species (cattle, sheep, goat, elk, and deer) in animal feeds has been developed. Primers targeting conserved mitochondrial DNA sequences amplified ruminant DNA in a universal PCR, and the digoxigenin-labeled amplicons were hybridized with an array of species-specific oligonucleotide capture probes on a polyester cloth support. The hybridized amplicons were detected on the cloth by sequential reactions with antidigoxigenin antibody–peroxidase conjugate and chromogenic substrate solution. This cloth-based hybridization array system provided sensitive and specific detection and identification of meat meal containing rendered cattle, sheep, goat, elk, and deer material blended in feeds.

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