Development of a 16S-23S rRNA intergenic spacer-based quantitative PCR assay for improved detection and enumeration ofLactococcus garvieae

Abstract

Lactococcus garvieae is an important foodborne pathogen causing lactococcosis associated with hemorrhagic septicemia in fish worldwide. A real-time quantitative polymerase chain reaction (qPCR) protocol targeting the 16S-23S rRNA intergenic spacer (ITS) region was developed for the detection and enum-eration of L. garvieae. The specificity was evaluated using genomic DNAs extracted from 66 cocci strains. Fourteen L. garvieae strains tested were positive, whereas 52 other strains including Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. hordniae and Lactococcus lactis ssp. cremoris did not show a specific signal. The minimal limit of detection was 2.63 fg of purified genomic DNA, equivalent to 1 genome of L. garvieae. The optimized protocol was applied for the survey of L. garvieae in naturally contaminated fish samples. Our results suggest that the qPCR protocol using ITS is a sensitive and efficient tool for the rapid detection and enumeration of L. garvieae in fish and fish-containing foods.

An update on the detection methods of Parachlamydia acanthamoebae, an atypical agent of pneumonia

“…It is suggested that PCR assays which target the internal transcribed spacer (ITS) region that links the 16S and 23S regions should be developed. Indeed, this has been employed before to detect and discriminate between various organisms such as Lactococcus garvieae, Streptococcus iniae, Vibrio spp., Campylobacter spp., Legionella spp, and Clostridium difficile (Berridge et al, 1998;Hoffmann et al, 2010;Mendoza et al, 1998;Riffard et al, 1998;Stubbs et al, 1999;Thanh et al, 2013). Targeting the ITS region for PCR has the inherent advantage of permitting intraspecies differentiation as species-species sequence polymorphisms can be detected easily because the ITS region varies greatly in size and sequence among closely related bacterial species in comparison with the more evolutionarily constrained 16S and 23S rRNA genes (Berridge et al, 1998;Hoffmann et al, 2010;Riffard et al, 1998).…”

“…Targeting the ITS region for PCR has the inherent advantage of permitting intraspecies differentiation as species-species sequence polymorphisms can be detected easily because the ITS region varies greatly in size and sequence among closely related bacterial species in comparison with the more evolutionarily constrained 16S and 23S rRNA genes (Berridge et al, 1998;Hoffmann et al, 2010;Riffard et al, 1998). Additionally, bacteria may have multiple alleles of the rRNA gene cluster and as such this would permit the differentiation of epidemiologically related and unrelated strains in addition to potentially increasing the sensitivity of the PCR assay (Kiss et al, 1977;Thanh et al, 2013). In the context of P. acanthamoebae, there is only one PCR assay available which targets the ITS region and even then the primers targeting the region were Chlamydiaceae order specific hence indicating that this avenue has not been widely explored yet (Lutz-Wohlgroth et al, 2006).…”

16S-23S rRNA Internal Transcribed Spacer Region (ITS) Sequencing: A Potential Molecular Diagnostic Tool for Differentiating Lactococcus garvieae and Lactococcus petauri

“…The amplification of a short region (290 bp) of ITS has already been suggested as a useful tool for L. garvieae diagnoses [ 19 ]. The ITS region has been considered a good tool for species specific bacterial identification among related organisms due to its genetic variability in size and sequence compared to 16S rRNA and 23S rRNA genes [ 20 ]. This method was recently used for molecular diagnosis in piscine lactococcosis [ 6 ]; however, its role in distinguishing between L. garvieae and L. petauri has not been reported.…”