Improved multiplex TaqMan qPCR assay with universal internal control offers reliable and accurate detection of Clavibacter michiganensis

Abstract

Aim: 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. Methods and Results: 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. Conclusion:The triplex TaqMan qPCR provides a specific and sensitive diagnostic assay for Cm. Significance and Impact of the Study: 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.

Development of a multiplex TaqMan qPCR targeting unique genomic regions for the specific and sensitive detection of Pectobacterium species and P. parmentieri

“…Therefore, we have included a universal exogenous internal positive control (UIC) to demonstrate the reliability of our multiplex TaqMan qPCR. Ramachandran et al (2021) designed a heterologous and exogenous fully synthetic DNA with its own primers set (UIC-Fwf/UIC-Rwf) and probe (UIC-P), which makes this UIC non-homologous to any natural target including plant host. The designed UIC was spiked with samples before PCR amplification.…”

“…All sets of primers and probes specific for either the genus Pectobacterium or P. parmentieri were designed with Primer3web (v. 4.1.0). Sense and anti-sense primer sequences targeting a non-homologous artificially synthesized DNA, used as UIC, were taken from Ramachandran et al (2021). In addition, customized AT-rich flap sequences were incorporated at the 5′ ends of each primer with the aim of optimizing the specificity and amplification yield as reported by Arif and Ochoa-Corona (2013) and Larrea-Sarmiento et al (2019).…”

“…Results of the nucleotide BLAST (BLASTn) analysis carried on 07/08/19. b Description of both primers and probe corresponding to the UIC were retrieved from Ramachandran et al (2021).…”

“…Considering the above background, the main purpose of this research was to develop a multiplex TaqMan qPCR for the specific detection of the genus Pectobacterium and P. parmentieri . We also added a universal internal control (UIC; Ramachandran et al, 2021) to enhance the reliability of our assay. Using comparative genomics approaches, we found unique genomic loci exclusively present only in the target genomes.…”

Adsorption of Recombinant Human β-Defensin 2 and Two Mutants on Mesoporous Silica Nanoparticles and Its Effect against Clavibacter michiganensis subsp. michiganensis

“…Cmm is a Gram-positive bacterium distributed worldwide that causes bacterial tomato canker disease, which can cause large economic losses to farmers if not treated properly and in a timely manner [ 4 ]. The use of contaminated seeds and asymptomatic transplants causes the propagation of the disease, detection methods are inefficient at differentiating Cmm from non-pathogenic Clavibacter strains [ 5 ]. However, the possibility exists to make detection methods more efficient by merging emerging and mature technologies.…”