Improved multiplex TaqMan qPCR assay with universal internal control offers reliable and accurate detection of Clavibacter michiganensis.
Clavibacter michiganensis
Gram-positive bacteria
detection
internal control
multiplex qPCR
Journal
Journal of applied microbiology
ISSN: 1365-2672
Titre abrégé: J Appl Microbiol
Pays: England
ID NLM: 9706280
Informations de publication
Date de publication:
Sep 2021
Sep 2021
Historique:
revised:
16
12
2020
received:
21
09
2020
accepted:
21
01
2021
pubmed:
24
1
2021
medline:
13
10
2021
entrez:
23
1
2021
Statut:
ppublish
Résumé
Clavibacter michiganensis (Cm) is a seed-borne plant pathogen that significantly reduces tomato production worldwide. Due to repeated outbreaks and rapid spread of the disease, seeds/transplants need to be certified free of the pathogen before planting. To this end, we developed a multiplex TaqMan qPCR assay that can accurately detect Cm in infected samples. A specific region of Cm (clvG gene) was selected for primer design using comparative genomics approach. A fully synthetic universal internal control (UIC) was also designed to detect PCR inhibitors and false-negative results in qPCRs. The Cm primers can be used alone or in a triplex TaqMan qPCR assay with UIC and previously described Clavibacter primers. The assay was specific for Cm and detected up to 10 fg of Cm DNA in sensitivity and spiked assays. Addition of the UIC did not change the specificity or sensitivity of the multiplex TaqMan qPCR assay. The triplex TaqMan qPCR provides a specific and sensitive diagnostic assay for Cm. This assay can be used for biosecurity surveillance, routine diagnostics, estimating bacterial titres in infected material and for epidemiological studies. The UIC is fully synthetic, efficiently amplified and multiplex compatible with any other qPCR assay.
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1405-1416Subventions
Organisme : USDA National Institute of Food and Agriculture
ID : 9038H
Organisme : National Science Foundation
ID : DBI-1561663
Informations de copyright
© 2021 The Society for Applied Microbiology.
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