RU2485177C1 - METHOD OF DETECTING RIFAMPICIN-RESISTANT ISOLATES Mycobacterium tuberculosis - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of detecting the mutations in rpoB gene by carrying out PCR is proposed, when in "real-time" the fragment of rpoB gene is created using primers Rpo11 (5'-GACGTCGAGGCGATCACAC-3') and Rpo12 (5'-GGCACGCTCACTTGACAGAC-3'), after that HRM-analysis is carried out. To increase the reliability of HRM-analysis of mutations in rpoB gene the equal amounts of tested DNA and DNA H37Rv are mixed. The concentration equalisation is carried out using quantitative PCR with use of the same pair of primers with creation of a calibration curve and use of regression equation.

EFFECT: method due to its high specificity and sensitivity enables to identify reliably and quickly the type of mutations in the rpoB gene associated with the emergence of resistance to rifampicin.

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Description

The present invention relates to molecular biology and medicine and can be used in the laboratory diagnosis of tuberculosis to quickly identify mutations in the rpoB gene associated with the formation of resistance to rifampicin.

Antibiotic resistance in mycobacteria of the tuberculosis complex is a fairly well-studied phenomenon in terms of molecular genetic mechanisms. Mutations associated with resistance to isoniazid, ethambutol, pyrazinamide, rifampicin and other anti-tuberculosis drugs have been described [1]. It was shown that 95% of rifampicin-resistant isolates have mutations in the “core” region of the rpoB gene encoding the 81-bp RNA polymerase β subunit. Most often, mutations occur in 531 (Ser-> Leu) and 526 (His-> Tyr) codons. Other nucleotide substitutions are also associated with the emergence of resistance to rifampicin: in 511, 515, 516, 526 and other codons, which are much less common.

Determining the resistance of mycobacteria by classical bacteriological methods takes 1-2 months, which can be considered "lost" for the patient. Automated systems using liquid media, which are based on the method of proportions, allow you to get the result within 14 days, while its cost is very high. Hybridization methods of PCR analysis of drug resistance give a result within 4-5 hours, are also costly and require additional equipment.

A number of methods have been developed to diagnose the drug resistance of mycobacteria to rifampicin by directly detecting mutations in the “core” region of the rpoB gene using various molecular genetic approaches [1]. Methods for the detection of drug resistance, based on the analysis of mycobacterial DNA, are performed within a few days, show sufficient specificity and sensitivity. However, these methods have a significant drawback, such as a high probability of contamination with a large flow of analyzes. With the advent of the real-time PCR method, this drawback can be overcome, while significantly reducing the analysis time. In addition, it is worth noting the simplicity of the method for staff.

In 1997, a new type of analysis of PCR products was proposed, based on the use of intercalating fluorescent DNA dyes and the capabilities of equipment for real-time PCR [2]. After standard PCR, the reaction mixture containing the amplified DNA fragment undergoes heating, which leads to the transition of the DNA fragment from the double-stranded form to single-stranded, dye dissociation and a decrease in fluorescence. The result is a characteristic melting curve specific for a particular PCR fragment. Attempts to increase the informativeness of fluorescence monitoring of DNA melting have led to the emergence of a method for analyzing melting curves with high resolution (HRM, HRMA, HRMCA) [3]. Mutations within the analyzed PCR DNA fragment lead to small changes in the melting temperature, which shifts the curve along the axis of the temperature value or, in some cases, changes its shape. The use of HRM analysis for the rapid detection of mutations causing drug resistance in tuberculosis mycobacteria has been previously described [4-6]. However, the described approaches do not allow achieving highly reproducible results, which is especially important in clinical practice.

Closest to the claimed method, the prototype is a typing method proposed by K.G. Hoek et al. [4], where HRM analysis was first used to detect mutations in the rpoB gene. It is known that when analyzing single nucleotide substitutions (SNPs) in the human genome by HRM, heterozygotes are most reliably detected. This is due to the fact that after melting of the amplicons of two types and their further reassociation, four types of molecules are formed - two initial and two bearing unpaired bases (mis matches) in the SNP region, which have a significantly lower melting point. The superposition of the melting curves of these DNA fragments gives a characteristic curve, which often contains two kinks and is significantly different from the melting curve of the wild-type amplicon. This fact has been used in a number of studies on screening highly penetrant mutations for human monogenic diseases [7]. In this paper, Hoek et al. the wild-type rpoB portion of the wild-type gene (H37Rv strain) was amplified and the analyzed clinical sample, then amplicons were mixed, another 10 rounds of PCR were performed, and already after that the analysis of the melting curves was carried out. The disadvantage of this method is the decrease in homogeneity and the high probability of cross-contamination with amplicons of any type.

The proposed method for detecting rifampicin-resistant M. tuberculosis isolates by determining mutations in the rpoB gene associated with the formation of rifampicin resistance consists in real-time PCR using HRM analysis, which differs in that “ artificial ”heteroduplexes due to the simultaneous co-amplification of wild-type DNA and test DNA, which can increase the reliability of the analysis. Primers of the following structure are used in PCR: Rpo11 5'-GACGTGGAGGCGATCACAC-3 'and Rpo12 5'-GGCACGCTCACTTGACAGAC-3'. In this case, the main principle of real-time PCR, namely the analysis of PCR products with a closed lid, is retained, which eliminates the possibility of contamination with amplicons obtained during the reaction.

The method is as follows

DNA isolation from M. tuberculosis culture is carried out according to the described procedure [8].

Amplification reactions are performed using a thermal cycler with an optical unit for fluorescence detection CFX96 (Bio-Rad). To record real-time fluorescence, SYTO9 intercalating dye is added to the reaction mixture. The quality of the amplification reaction is considered acceptable when the following conditions are met:

- the efficiency of the amplification reaction is more than 80% (E> 0.8);

- the correlation coefficient between the expected and empirical equations of the calibration curve is greater than 0.99 (r ² >99);

- the difference between Ct values for repeating points of the calibration curve does not exceed 0.5 cycle;

- lack of non-specific amplification products.

PCR efficiency was calculated using equation (1)

log (R ₀ ) = log (R _N ) -log (1 + E) xN _t ,

Where

- N is an independent variable corresponding to the number of the amplification cycle on which measurements are made;

- R _N is the number of amplified molecules after the Nth amplification cycle (directly proportional to the fluorescence value of the amplification mixture);

- R ₀ is the initial number of amplified molecules;

- E - amplification efficiency.

For this, samples of the calibration curve (four consecutive 4-fold dilutions of the analyzed and reference (H37Rv) DNA) are amplified with a pair of primers (Rpo11 / Rpo12). Using the calibration direct and regression equation (1), the concentrations of the analyzed DNA samples are equalized to the concentration of the least concentrated DNA.

Directly real-time PCR followed by HRM analysis is carried out by mixing an equal amount of the tested DNA and H37Rv DNA. PCR is carried out in a volume of 20 μl containing 67 mM Tris-HCl (pH 8.9), 16 mM ammonium sulfate, 1.5 mM MgCl2, 0.01% Tween 20, 0.2 mM dNTP, 0.5 μM oligonucleotide solutions primers (Rpo11 5'-GACGTGGAGGCGATCACAC-3 'and Rpo12 5'-GGCACGCTCACTTGACAGAC-3') and 1 U / act Taq polymerase. The reaction is carried out on a CFX96 thermocycler (Bio-Rad, USA) with initial denaturation at 96 ° C for 2 min, then 35 cycles with denaturation at 95 ° C for 5 s, annealing the primers at 63 ° C for 5 s and synthesis at 72 ° C for 5 s.

To analyze the PCR melting curve, the mixture is heated to 96 ° C for 5 min, then incubated for a minute at a temperature of 63 ° C and melting is carried out at a rate of 0.2 ° C for one step of taking a fluorescent signal, up to 96 ° C. To assess the quality of the PCR aliquots of the reaction fractionated in 6% polyacrylamide gel. For HRM analysis, Precision Melt AnalysisTM Software (Bio-Rad, USA) is used. The specificity of the amplification reaction is confirmed by the absence of additional peaks on the melting curve and additional bands on the electrophoregram.

Typical melting curves are shown in Figures 1 and 2. A graphical representation of the melting curves of amplification products of the rpoB gene region is presented. The replacement of the rpoB gene in codon 531 TCG-> TTG horizontally shifts the peaks of the melting curves to the left due to a decrease in the melting temperature (see Fig. 1). The use of intercalating dye SYBRGreen I (cheaper) reliably detected this mutation. The nucleotide substitution in codon 526 САС-> СТС in the rpoB gene changes the nature of the melting curves, forming a characteristic kink (see Fig. 2), this mutation is detected using SYTO9

Thus, the proposed approach has high specificity and sensitivity in the direct analysis of DNA isolated from sputum. Screening for rifampicin resistance using our approach, given its speed and low cost, can be effective in detecting MDR tuberculosis.

The developed method for detecting mutations in the rpoB gene of Mycobacterium tuberculosis has high sensitivity and specificity (100%), as well as the lowest cost among the alternative approaches described to date, has a high level of reliability and reproducibility. The method was tested on a representative sample of 200 M. tuberculosis DNA samples. The total analysis time, starting with a biological sample (bacterial cells or sputum), did not exceed 5.5 hours. It is worth noting that the developed approach to the analysis of point mutations can be suitable for the analysis of other nucleotide substitutions of scientific and practical interest. The developed method is well adapted to the instrumental capabilities of modern clinical diagnostic laboratories and, thus, it can now be used in phthisiatric practice.

Claims (1)

A method for detecting rifampicin-resistant Mycobacterium tuberculosis isolates by determining mutations in the rpoB gene associated with the formation of rifampicin resistance by real-time PCR using HRM analysis, characterized in that amplification of the mixture with an equal amount of the tested DNA and Wild type DNA using primers
Rpo11: 5'-GACGTGGAGGCGATCACAC-3 ';
Rpo12: 5'-GGCACGCTCACTTGACAGAC-3 ',
heteroduplexes are formed due to the simultaneous co-amplification of wild-type DNA and the test DNA, and at the first stage of PCR, the concentration is equalized using quantitative PCR using the same pair of primers (Rpo11 / Rpo12) using a calibration curve and a regression equation.

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