Abstract
The vaginal microbiota balance is quite fragile and susceptible to the development of vaginosis and candidiasis. The current diagnostic method for bacterial vaginosis relies on the evaluation of different bacterial morphotypes using the Nugent score. This method is only partially in correlation with a DNA sequencing-based diagnostic or Amsel criteria used by clinicians, suggesting the need for new molecular approaches dedicated to the diagnosis of BV. The objective of this study was to develop and validate a quantitative polymerase chain reaction (qPCR) assay for the specific and rapid detection of three vaginal pathogens, i.e. Candida, Gardnerella and Atopobium and the commensal Lactobacillus genera. For this purpose, four oligonucleotide primer pairs were designed and tested to obtain optimal amplification of the four target genera. The qPCR assay was also tested on the non-target genera and on human DNA. The designed primers allowed specific amplification of the target organisms in vitro and in clinical vaginal samples. The qPCR assay designed in this study is effective to specifically detect these genera in clinical samples as a molecular technique complementary to the Nugent score. It can be used in epidemiological studies for understanding the role of these pathogens and to follow their abundance in the microbiota in disease processes such as the development of vulvovaginal candidiasis and bacterial vaginosis.
Highlights
Lactobacillus spp. are a major component of the vaginal microbiota [1, 2] These bacteria play an important role in preventing colonization by undesirable organisms by exclusion, modulation of host immune response or production of various antibacterial compounds such as lactic acid or antimicrobial peptides [3, 4]
We designed primers that target the three major vaginal genera associated with bacterial vaginosis and non-optimal vaginal microbiota as well as Lactobacillus, which is associated with optimal vaginal microbiota
The targeted genes were the 16S rRNA gene for the genera Lactobacillus and Gardnerella, the 18S rRNA gene for Candida, and the Ribonuclease P encoding gene for the genus Atopobium (Table 1). Their specificity has been checked using DNA extracted from 12 Lactobacillus spp., four Candida, one Gardnerella and one Atopobium strains from our culture collection (Table 2) and from human Caco-2 cells
Summary
Lactobacillus spp. are a major component of the vaginal microbiota [1, 2] These bacteria play an important role in preventing colonization by undesirable organisms by exclusion, modulation of host immune response or production of various antibacterial compounds such as lactic acid or antimicrobial peptides [3, 4]. The current diagnostic method for BV in research setting relies on the evaluation of different bacterial morphotypes on a Gram-stained vaginal smear, known as the Nugent score [14] This method fails to differentiate a normal from an abnormal vaginal microbiota in a significant proportion of women, suggesting the need for new molecular approaches dedicated to the diagnosis of BV. Quantitative PCR technology represents a precise, quantitative, more affordable and faster than sequencing-based approaches that could be used in research and clinical trials Such test would allow the rapid detection of early signs of transition towards BV state as well as BV remission after preventive and curative therapies, including the live biotherapeutic products (LBP). These primers were checked for specificity against DNA extracted from in vitro microbial cultures (single strain or coculture) as well as DNA extracted from human vaginal samples collected as part of the UMB-HMP study and for which the composition of the vaginal microbiota had already been established [16]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: International Journal of Microbiology and Biotechnology
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
