RU2486255C1 - OLIGONUCLEOTIDES AND METHOD OF DETERMINING DNA OF BACTERIA BELONGING TO FAMILY Chlamydiaceae - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of detecting DNA of microorganisms belonging to the family Chlamydiaceae is proposed, by the method of PCR with use of primers and a probe, complementary to conserved region of the 16S ribosomal riziform gene.

EFFECT: increased efficiency of using the composition.

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Description

The present invention relates to the field of molecular biology. In particular, it relates to methods and reagents for detecting microorganisms belonging to the Chlamydiaceae family. The scope of the invention is medicine, medical microbiology, veterinary medicine, biology, molecular biology.

Chlamydiae are obligate intracellular parasites that cause a wide range of diseases in humans, animals and birds. According to the current classification, 8 families are assigned to the Chlamydiae phylum, however, the microorganisms from the Chlamydiaceae family, in which there are 9 species, divided into two genera, Chlamydia and Chlamydophila, are the most famous and relevant in medical and veterinary importance. The genus Chlamydia includes C.trachomatis, C.muridarum, C.suis. The genus Chlamydophila includes C. pneumonia, C. abortus, C. caviae, C. felis, C. psittaci, C. pecorum. Chlamydia is characterized by a large polymorphism of clinical manifestations and the absence of specific symptoms. In humans, chlamydia is the causative agent of trachoma, venereal lymphogranuloma, diseases of the urogenital and respiratory tract, conjunctivitis, zoo and ornithanthroponous diseases. With some degree of probability, these bacteria are associated with the development of cardiovascular pathologies, atherosclerosis, Alzheimer's disease, some forms of arthritis and bronchial asthma. In animals, chlamydia can cause respiratory diseases and abortion reactions in cattle and small cattle, conjunctivitis and rhinitis in domestic animals, a wide range of diseases in ungulate animals, including pigs, systemic diseases in parrots, poultry, etc. However, the true role chlamydia in the pathogenesis of many diseases remains unclear, including due to the lack of simple and reliable means of detection, identification and differentiation of chlamydia, taking into account the current level of knowledge about their biological m variety.

Diagnosis of chlamydial infections is a difficult task due to both an extremely wide range of clinical manifestations and the disadvantages of the traditionally used diagnostic methods. The most powerful approach for the diagnosis of bacterial and viral infections in general, and chlamydial in particular, is the polymerase chain reaction (PCR) method, which is based on the multiple copying of a specific DNA or RNA fragment using the DNA polymerase enzyme, which is a marker for this type of infectious pathogen diseases. Currently, PCR is the most sensitive way to indicate microorganisms, capable of potentially detecting single particles of pathogens of infectious diseases. The advantages of this type of diagnosis are also a direct determination of the pathogen itself, and not of proteins or other products of its vital activity, high specificity, high speed of obtaining the result, as well as the ability to diagnose chronic and latent infections, which is especially important in the case of chlamydia. There are several varieties of PCR described, for example, in: PCR Protocols: A guide to methods and applications, 1990 (ed. M.A. Innis et al.), Academic Press, San Diego, CA; PCR Strategies, 1995 (ed. M. A. Innis et al.), Academic Press, San Diego, CA; PCR Protocols. Series: Methods in Molecular Biology, 2003, Vol. 226 (eds., J. M. S. Bartlett, D. Stirling), Humana Press. In earlier PCR versions, gel electrophoresis was used to detect amplification products, which complicates the analysis. Modern modifications of the method use fluorescent labels and detection of a fluorescent signal directly during the reaction, for which the method is often called real-time PCR ("real-time PCR"). Another name for the method is quantitative PCR (“quantitative PCR, qPCR”), which reflects the capabilities of the method to quantify the detected object.

A known method for detecting bacteria belonging to the family Chlamydiaceae (with fluorescence detection of amplification products), in which the gene 23 S rRNA and the so-called FRET probes were used as a target (DeGraves et al. 2003). The authors of the method did not take into account the 9 species classification of chlamydia, which came into use at the beginning of the 20th century (Everett et al. 1999), and declared the detection of only three species of C. psittaci, C. corum and C. pneumonia.

Another method is described in (Robertson et al. 2009). In this method, the 16S rRNA gene was used as a target, and the fluorescent signal was generated as a result of using the intercalating dye SYTO 9 green. The disadvantage of methods with intercalating dyes is their lower specificity, since the dye will also interact with non-specific amplicons, giving rise to a fluorescent signal.

Closest to the present invention is the method described in (Wooters et al. 2009). The 16S rRNA gene and Taqman probe were used as a target. The amplicon size was 208 bp, which is not optimal for this method, because a significant part of the target is amplified, which does not bind to the probe and is unnecessary.

An object of the invention is to identify the DNA of any of 9 species of bacteria belonging to the Chlamydiaceae family in the studied material with a high degree of specificity and with the detection of reaction results in real time using a family-specific probe.

The specified technical result is achieved in that in the method for determining the DNA of microorganisms belonging to the Chlamydiaceae family, a polymerase chain reaction is used using two primers and one probe, characterized in that oligonucleotides of the formula Seq1 and Seq2 are used as primers, and an oligonucleotide is used as a probe Seq3 formulas.

To select primers and a probe, a computer analysis of the nucleotide sequences of the 16S rRNA gene was performed; conservative regions of all species of the Chlamydiaceae family were determined within which flanking primers and probe could be selected. As a result, oligonucleotides were selected as follows

Seq1 5′-AAGAAGGGGATCTTAGGACCTTTCGGTT-3 ′

Seq2 5′-GCAGTGTCTCAGTCCCAGTGTTGGC-3 ′

Seq3 5′-TGACGTCTAGGCGGATTGAGAGATTG-3 ′

The calculated PCR product length for primers Seq1 and Seq2 is approximately 150 bp

The technical result of the claimed invention is a combination of the nucleotide sequences of two primers and a fluorescent probe, which can be used for real-time PCR to determine the DNA of microorganisms belonging to the Chlamydiaceae family.

The invention is illustrated by the following examples.

Example 1. Amplification of the 16S rRNA gene region of three types of chlamydia: C.trachomatis, C. pneumoniae, C. psittaci.

The performance of the primers was investigated in PCR with electrophoretic analysis of the reaction products.

The polymerase chain reaction was carried out on a DT-96 thermocycler, manufacturer DNA-technology, Moscow. As a matrix, DNA samples of C.trachomatis, C. pneumoniae, C.psittaci obtained as described in (Demkin, Zimin 2005) were used. As negative samples, mQ H ₂ O and DNA isolated from urogenital smears of healthy people were used.

The reaction was carried out in the following mode: 1 cycle of the preliminary denaturation stage 2 minutes at 95 ° C and 40 cycles, including 10 seconds at 95 ° C and 30 seconds at 55 ° C of the annealing stage. The analysis of amplification products was carried out by electrophoresis in a 2% agarose gel with ethidium bromide. The experimental results shown in Figure 1 show that only samples containing chlamydial DNA produce amplicons of the calculated size.

Example 2. Determination of DNA of C.trachomatis, C.pneumoniae, C.psittaci with fluorescence detection.

The example demonstrates the operability of the Seq3 probe in the detection mode of a fluorescent signal. The probe at the 5′-end contained FAM fluorescent dye, and the BHQ2 fluorescent quencher at the 3′-end.

The incubation mixture was as in Example 1, with the exception that a probe was added at a concentration of 0.17 μM. The set of samples studied and the amplification mode are the same as in Example 1, with the only difference being that the fluorescent signal was detected via the FAM channel at the stage of primer annealing at 55 ° C. Figure 2 shows the experimental results. The flare-up of a fluorescent signal is observed only in samples containing Chlamydia DNA. The flare-up of the fluorescent signal in the samples, where mQ H ₂ O was used as the matrix, and in the DNA sample isolated from the urogenital smear of a healthy person, were not detected.

The results obtained indicate that the selected oligonucleotides provide a specific definition of chlamydial DNA. The target sequences of those species of the Chlamydiaceae family that were not tested in the described experiments are fully complementary to the developed primers and probe, which ensures the universal operation of primers and probes in determining the DNA of any species of the Chlamydiaceae family.

BIBLIOGRAPHY

PCR Strategies, 1995 (ed. M. A. Innis et al.), Academic Press, San Diego, CA;

PCR Protocols. Series: Methods in Molecular Biology, 2003, Vol. 226 (eds., J. M. S. Bartlett, D. Stirling), Humana Press.

DeGraves FJ, Gao D, Hehnen HR, Schlapp T, Kaltenboeck B Quantitative detection of Chlamydia psittaci and C pecorum by high-sensitivity real-time PCR reveals high prevalence of vaginal infection in cattle. J Clin Microbiol 2003 Vol.41 (4): 1726-1729

Robertson T, Bibby S, O′Rourke D, Belfiore T, Lambie H, Noormohammadi AH.

Characterization of Chlamydiaceae species using PCR and high resolution melt curve analysis of the 16S rRNA gene. J Appl Microbiol. 2009 Vol. 107 (6): 2017-2028.

Wooters MA, Kaufhold RM, Field JA, Indrawati L, Heinrichs JH, Smith JG. A real-time quantitative polymerase chain reaction assay for the detection of Chlamydia in the mouse genital

tract model. Diagn Microbiol Infect Dis. 2009 Vol. 63 (2): 140-147.

Demkin, V.V., Zimin A.L. A new amplification target for PCR-RFLP detection and identification of Chlamydiaceae species. Arch Microbiol. 2005 Vol. 183 (3): 169-175.

Claims (2)

1. A method for determining bacteria of the Chlamydiaceae family, comprising the amplification of a bacterial nucleic acid in a test material by polymerase chain reaction using oligonucleotide primers with sequences of at least 75% homologous to the sequences of SEQ ID NO: 1 and SEQ ID NO: 2, and the probe with a sequence of not less than 75% homologous sequence of SEQ ID NO: 3 containing at the 5 ′ and / or 3 ′ ends of the label. 2. The method according to claim 1, where the labels of the probe can be radioisotopes, enzymes, fluorescent dyes, luminescent dyes, fluorescence-absorbing substances, biotin.

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