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.

Specific PCR Detection of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii, and Arcobacter cibarius in Chicken Meat

2009 ◽  
Vol 72 (7) ◽  
pp. 1491-1495 ◽  
Author(s):  
DANIELA PENTIMALLI ◽  
NICOLETTE PEGELS ◽  
TERESA GARCÍA ◽  
ROSARIO MARTÍN ◽  
ISABEL GONZÁLEZ
Keyword(s):  
Pcr Amplification ◽  
Chicken Meat ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Gyra Gene ◽  
Specific Primers ◽  
Specific Pcr ◽  
Arcobacter Butzleri ◽  
Species Specific

An enrichment PCR assay using species-specific primers was developed for the detection of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii, and Arcobacter cibarius in chicken meat. Primers for A. cryaerophilus, A. skirrowii, and A. cibarius were designed based on the gyrA gene to amplify nucleic acid fragments of 212, 257, and 145 bp, respectively. The A. butzleri–specific primers were designed flanking a 203-bp DNA fragment in the 16S rRNA gene. The specificity of the four primer pairs was assessed by PCR analysis of DNA from a panel of Arcobacter species, related Campylobacter, Helicobacter species, and other food bacteria. The applicability of the method was then validated by testing 42 fresh retail-purchased chicken samples in the PCR assay. An 18-h selective preenrichment step followed by PCR amplification with the four Arcobacter primer sets revealed the presence of Arcobacter spp. in 85.7% of the retail chicken samples analyzed. A. butzleri was the only species present in 50% of the samples, and 35.7% of the samples were positive for both A. butzleri and A. cryaerophilus. A. skirrowii and A. cibarius were not detected in any of the chicken samples analyzed. The enrichment PCR assay developed is a specific and rapid alternative for the survey of Arcobacter contamination in meat.

scholarly journals A novel subspecies-specific primer targeting the gyrase B gene for the detection of Pectobacterium carotovorum subsp. brasiliense

2019 ◽  
Vol 20 (10) ◽  
Author(s):  
Tri Joko ◽  
ALAN SOFFAN ◽  
MUHAMMAD SAIFUR ROHMAN
Keyword(s):  
Sequence Data ◽  
Soft Rot ◽  
Pectobacterium Carotovorum ◽  
Specific Primer ◽  
Base Pairs ◽  
Detection And Identification ◽  
Wide Range ◽  
Carotovorum Subsp ◽  
Rot Disease ◽  
The Tropics

Abstract. Joko T, Soffan A, Muhammad Saifur Rohman MS. 2019. A novel subspecies-specific primer targeting the gyrase B gene for the detection of Pectobacterium carotovorum subsp. brasiliense. Biodiversitas 20: 3042-3048. Pectobacterium carotovorum subsp. brasiliense is one of the major causative bacterial pathogens of the soft rot disease in various crops. It has a high virulence and a wide range of hosts in the tropics and the subtropics. Most often, conventional methods are not able to accurately distinguish P. carotovorum subsp. brasiliense from other subspecies. Thus, this study aimed to design a specific gyrase B gene (gyrB) -based primers for the detection and identification of soft rot pathogen. The specific primers design was based on the alignment using gyrB gene sequence data from P. carotovorum subsp. brasiliense and other data from the GenBank. The primers comprised of F-gyr-Pcb (5’-CAC AGG CAC CGC TGG CTG TT-3’) and R-gyr-Pcb (5’-CGT CGT TCC ACT GCA ATG CCA-3’) with an amplicon of 336 base pairs. The specificity of the primers pair was verified both in silico and in polymerase chain reaction (PCR) assays, where the primers could only detect P. carotovorum subsp. brasiliense. Primers’ sensitivity was determined by qualitative PCR with a detection limit of less than 0.5 ng/µL of genomic DNA. Hence, the proposed detection tool can be beneficial to advance further studies on the ecology and epidemiology of soft rot diseases.

scholarly journals A targeted approach with nanopore sequencing for the universal detection and identification of flaviviruses

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Patrick Reteng ◽  
Linh Nguyen Thuy ◽  
Tam Tran Thi Minh ◽  
Maria Angélica Monteiro de Mello Mares-Guia ◽  
Maria Celeste Torres ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Detection System ◽  
Diagnostic System ◽  
Clinical Samples ◽  
Species Differentiation ◽  
Nanopore Sequencing ◽  
Bioinformatics Pipeline ◽  
Detection And Identification ◽  
Universal Detection ◽  
Species Specific

AbstractNucleic acid test (NAT), most typically quantitative PCR, is one of the standard methods for species specific flavivirus diagnosis. Semi-comprehensive NATs such as pan-flavivirus PCR which covers genus Flavivirus are also available; however, further specification by sequencing is required for species level differentiation. In this study, a semi-comprehensive detection system that allows species differentiation of flaviviruses was developed by integration of the pan-flavivirus PCR and Nanopore sequencing. In addition, a multiplexing method was established by adding index sequences through the PCR with a streamlined bioinformatics pipeline. This enables defining cut-off values for observed read counts. In the laboratory setting, this approach allowed the detection of up to nine different flaviviruses. Using clinical samples collected in Vietnam and Brazil, seven different flaviviruses were also detected. When compared to a commercial NAT, the sensitivity and specificity of our system were 66.7% and 95.4%, respectively. Conversely, when compared to our system, the sensitivity and specificity of the commercial NAT were 57.1% and 96.9%, respectively. In addition, Nanopore sequencing detected more positive samples (n = 8) compared to the commercial NAT (n = 6). Collectively, our study has established a semi-comprehensive sequencing-based diagnostic system for the detection of flaviviruses at extremely affordable costs, considerable sensitivity, and only requires simple experimental methods.

Molecular identification of three of the most important mealybug species (Hemiptera: Sternorrhyncha: Coccoidea: Pseudococcidae) on ornamental plants in Guilan province, Iran

Zootaxa ◽  
2011 ◽  
Vol 3009 (1) ◽  
pp. 46 ◽  
Author(s):  
REZA HOSSEINI ◽  
JALIL HAJIZADEH
Keyword(s):  
Ornamental Plants ◽  
Accurate Method ◽  
Planococcus Citri ◽  
Morphological Identification ◽  
Specific Primers ◽  
Pseudococcus Viburni ◽  
Species Specific ◽  
Pseudococcus Comstocki ◽  
Guilan Province ◽  
Study Species

Mealybugs (Coccoidea: Pseudococcidae) are serious pests, particularly as invasive species on many agricultural products. Morphological identification of mealybugs is based on adult female characters that, in the absence of adult females or with damaged specimens, can be problematic, especially when identification is required urgently, such as that involving the exportation/importation market. In this study, species-specific primers were designed to identify three of the most abundant mealybug species found on ornamental plants in Guilan province, Iran: Planococcus citri (Risso), Pseudococcus viburni (Signoret) and Pseudococcus comstocki (Kuwana). By generating amplification products of different sizes, the three species-specific primers, along with universal COI primers, were successfully used in multiplex PCR tests to identify all three mealybug species in a single reaction. Analysis of a large array of specimens from different geographic locations on different host plants showed that this was a reliable and accurate method.

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 Pratylenchus neglectus (Nematoda: Pratylenchidae) under the rhizosphere of Brassica napus

2012 ◽  
Vol 49 (2) ◽  
pp. 92-95 ◽  
Author(s):  
S. Kumari
Keyword(s):  
Brassica Napus ◽  
Ribosomal Dna ◽  
Soil Samples ◽  
Morphological Features ◽  
Morphological Identification ◽  
Specific Primers ◽  
Pratylenchus Neglectus ◽  
Species Specific

AbstractSoil samples under the rhizosphere of Brasicca napus were collected from three localities (Bílé Podolí, Prague, Kylešovice). Two localities Prague and Kylešovice were positive for the presence of Pratylenchus neglectus. The species was identified using morphological features and the morphological identification was verified by using published species-specific primers and by sequencing 18S and 28S genes of ribosomal DNA.

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.

Molecular and morphological discrimination betweenTylospora fibrillosaandTylospora asterophoramycorrhizae

1999 ◽  
Vol 77 (1) ◽  
pp. 11-21 ◽  
Author(s):  
Ursula Eberhardt ◽  
Lutz Walter ◽  
Ingrid Kottke
Keyword(s):  
Fragment Length Polymorphism ◽  
Pcr Primers ◽  
Morphological Identification ◽  
Chain Reaction ◽  
Specific Primers ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Species Specific ◽  
First Time

Among the mycorrhizal types of spruce, Tylospora-type mycorrhizae are the most constant and abundant. Two species of the genus Tylospora occur in Europe, Tylospora fibrillosa and Tylospora asterophora. Mycorrhizae of T. asterophora are described in detail for the first time. Sequences of the internal transcribed spacer (ITS) of the ribosomal genes were obtained from T. fibrillosa and T. asterophora mycorrhizae, sporocarps, and cultured mycelium. Discrimination and identification of the two species by ITS polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) are discussed in the light of inter- and intra-specific variability. Species-specific PCR primers were designed to distinguish both species. Molecular screening of Tylospora-type mycorrhizae from field material led to unambiguous results, whereas morphological identification is likely to fail because of great similarity even at the microscopic level.Key words: Tylospora asterophora, Tylospora fibrillosa, ectomycorrhizae, taxon specific primers (TSOPs), ITS sequences.

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