A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac

Autor: Jinbo Peng, Hao Liu, Tom Hsiang, Lu Zheng, Junbin Huang, Miaomiao Sun, Ya Zhang, Fuhua Fei
Jazyk: angličtina
Rok vydání: 2019
Předmět:
0106 biological sciences
0301 basic medicine
medicine.medical_specialty
soft-rot bacteria
Pectobacterium
Loop-mediated isothermal amplification
comparative genomics
01 natural sciences
Sensitivity and Specificity
Catalysis
Article
lcsh:Chemistry
Inorganic Chemistry
03 medical and health sciences
Bacterial soft rot
Pectobacterium aroidearum
Molecular genetics
medicine
Physical and Theoretical Chemistry
lcsh:QH301-705.5
Molecular Biology
Gene
Spectroscopy
Soil Microbiology
Plant Diseases
Comparative genomics
biology
Organic Chemistry
General Medicine
biology.organism_classification
Molecular biology
Computer Science Applications
genomic DNA
030104 developmental biology
lcsh:Biology (General)
lcsh:QD1-999
Genes
Bacterial
GenBank
konjac
Rhizosphere
Primer (molecular biology)
loop-mediated isothermal amplification
Nucleic Acid Amplification Techniques
Amorphophallus
010606 plant biology & botany
Zdroj: International Journal of Molecular Sciences
International Journal of Molecular Sciences, Vol 20, Iss 8, p 1937 (2019)
Volume 20
Issue 8
ISSN: 1422-0067
Popis: Bacterial soft rot caused by Pectobacterium species is a serious disease in konjac (Amorphophallus konjac), a healthy source of starch particularly in East Asia. An effective diagnostic method is crucial to control the disease and reduce losses in konjac production. In this study, we evaluated a loop-mediated isothermal amplification (LAMP) assay with a specific primer set for the rapid and accurate detection of P. aroidearum. A comparative genomics approach was used to identify the specific genes suitable for the design of LAMP primers. The candidate target genes were determined through a first-round comparison with a 50-genome nucleotide database, and subjected to a second-round screening with the GenBank NR database. As a result, nine specific genes of P. aroidearum were selected for LAMP primer design. After screening of the primers, the primer set 1675-1 was chosen for LAMP detection owing to its high specificity and sensitivity. The LAMP assay could detect the presence of P. aroidearum genomic DNA at a concentration as low as 50 fg and 1.2 ×
104 CFU/g artificially infected soil within 40 min at 65 °
C. Subsequently, this primer set was successfully used to specifically detect P. aroidearum in naturally infected and non-symptomatic plant samples or soil samples from the field. This study indicates that a comparative genomic approach may facilitate the development of highly specific primers for LAMP assays, and a LAMP diagnostic assay with the specific primer set 1675-1 should contribute to the rapid and accurate detection of soft-rot disease in konjac at an early stage.
Databáze: OpenAIRE
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