Biomedical Chemistry: Research and Methods 2019, 2(2), e00084

The qPCR analysis of vaginal microflora for the diagnosis of bacterial vaginosis

Senina M.Е.1*, Savochkina Y.А.1, Skvortsov L.V.1, Popova T.I.2, Dzhedzheia L.V.2

1«Lytech» Ltd, 3A/2 Malaya Semyonovskaya str., Moscow, 107023 Russia; *e-mail: senina_maria@mail.ru
2«Lytech Laboratory» Ltd, 3A/2 Malaya Semyonovskaya str., Moscow, 107023 Russia

Keywords: real-time PCR; Eggerthella; Bacterial Vaginosis–Associated Bacteria ;Megasphaera type 1; Femoscreen

DOI: 10.18097/BMCRM00084

The whole version of this paper is available in Russian.

The correct information about of the vaginal microflora plays an important role in preventing the occurrence of urinary tract infections and sexually transmitted infections among women. Disbalance of obligate and facultative microflora causes disbacteriosis, a risk factor for emergence of infectious diseases. It is known that the cause of bacterial vaginosis (BV) is not a single pathogen but a impairments in of the general balance of the vaginal microflora, which manifests a decrease of the normal microflora (Lactobacillus spp) and intense increase of pathogenic aerobic and anaerobic bacteria. The development of molecular genetic analysis methods, in particular, approaches based on the use of polymerase chain reaction (PCR), significantly expanded understanding of the diversity of microbial biotopes, including identification of the key and new «players» in the development of BV. The aim of our study was3 to evaluate the performance of real-time PCR kit «Femoscreen» («Lytech», Russia) for comprehensive BV diagnosis.
Figure 1. Scheme of analysis with the «Femoscreen» reagent kit.

Registration of amplification products come off an amplification reaction is made at each amplification cycle in real time. A number of an amplification cycle where the fluorescence curve intersected a border line (Ct) is automatically calculated by analyzing program using specific tuning values. The value of the Ct is negatively related to a concentration of DNA in a sample. For quantitative evaluation, Ct value (a number of an amplification cycle where the fluorescence curve intersected a border line) of sample being examined is compared to a Ct value of a PCS (Positive Control Sample) with a known DNA concentration (Fig. 2). DNA concentration in an analyzed sample is calculated according to the formula:

$${{\rm C}}_{{\rm Sample}}{\rm =\ }{{\rm C}}_{{\rm PCS}}\times {\rm \ }{{\rm 2}}^{{\rm (}{{\rm Ct}}_{{\rm PCS}}{\rm -}{{\rm Ct}}_{{\rm Sample}}{\rm )}}$$
(1),
where CSample - concentration of a DNA of a disease originator (or a human) in a sample being examined, expressed in genome equivalents/ml (GE/ml); CPSC - concentration of a plasmid caring a specific DNA fragment of a disease originator (or a human) in PCS, expressed in genome equivalents/ml (presented in a leaflet with a diagnostic set); CtSample - a number of an amplification cycle where the fluorescence curve of a sample being examined intersected a border line ; CtPCS - a number of an amplification cycle where the fluorescence curve of a positive control sample intersected a border line.
Figure 2. Amplification curves and Ct determination.
Figure 3. Examples of the interpretation for the samples referred to the group «normal bacterial vaginal flora» (A), «altered vaginal flora» (B) and «bacterial vaginosis»(C).

CLOSE
Table 1. The Nugent scoring system (from 0 to 10) for diagnosis of bacterial vaginosis by microscopy.

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Table 2. The obtained values of diagnostic sensitivity and specificity of the «Femoscreen» reagent kit.

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