A method for the diagnosis of chlamydial infection (options)

 

(57) Abstract:

The invention relates to biotechnology, biology, medicine and veterinary medicine. Developed three methods of detection of members of the family Chlamydiaceae, which consists in the detection of DNA of chlamydia by polymerase chain reaction (PCR) using oligonucleotide primers. Two ways to carry out one-step PCR using the first primer

5'-CAAACTCATAGACGAG(CT)AGT-3' (I)

and 5'-CTTTCAATGTTACAGAAAACTCTACAG-3' (II)

in the second method primer

5'-ATGCAAAATTACTGT(AT)TGGGTAAA-3' (III)

and 5'-CTTTCAATGTTACAGAAAACTCTACAG-3' (II).

Under the third method, the reaction is carried out in two stages: first using primers

5'-CAAACTCATCAGACGAG(CT)AGT-3' (I)

and 5'-CTTTCAATGTTACAGAAAACTCTACAG-3' (II)

and the second using primers

5'-CTTTCAATGTTACAGAAAACTCTACAG-3' (II)

and 5'-ATGCAAAATTATGT(AT)TGGGTAAA-3' (III).

At each stage hold 32 rounds of polymerase chain reaction, which include one cycle stage of denaturation of 4 min at 95oAnd stage of annealing, 2 min at 60oC, 30 cycles stage of elongation 1 min at 72oWith that stage of denaturation for 40 s at 95oWith stage and annealing 30 s at 60oC and one cycle stage 4 min elongation at 72oC. Use The, is Oceania species and presence related to chlamydia species. 3 S. and 3 C.p. f-crystals.

Group of inventions relates to biotechnology, may find application in medicine, medical Microbiology, biology, veterinary medicine and relates to a method of detecting chlamydia.

Chlamydia is widespread obligate intracellular parasites that cause a wide range of serious diseases of humans and animals. Chlamydia is able to exist in the body in the persistent form without any clinical symptoms, but have the ability to spontaneous activation, especially in cases of weakening of the immune status of the person. Currently accepted taxonomic system that separates the 9 species of chlamydia. Among them are particularly important for medicine importance of Chlamydia trachomatis, which is the infectious agent of blinding trachoma, urogenital abnormalities, OFTAL newborns and lymphogranuloma venerum, and Chlamydophila pneumoniae, which, according to the latest data, the cause of more than half of all pneumonias in the world, and presumably associated with a number of cardiovascular pathologies (in particular, ischemic heart disease, atherosclerosis, Alzheimer's disease and asthma.

That the ila psittaci (the causative agent of a wide range of ornithosis), Chlamydophila abortus (the most common cause of abortive reactions in cattle), Chlamydophila felis (the infectious agent of conjunctivitis and rhinitis domestic cats) and Chlamydophila pecorum (the causative agent of a wide range of diseases of ungulates, including pigs).

For the other three species of Chlamydia muridarum (the infectious agent of pneumonia mice), Chlamydia suis (the causative agent of several diseases of the European wild pig) and Chlamydophila caviae (causes diseases of the mucous epithelium of Guinea pigs) no recorded cases of human infection.

Detection of chlamydia is a difficult task because of the very wide spectrum of clinical manifestations and disadvantages of commonly used detection methods. A promising approach for the detection of chlamydia is DNA diagnostics based on polymerase chain reaction (PCR). Currently, PCR is the most sensitive method for the display of microorganisms with the potential to detect a single bacterial particles. The advantages of this type of detection are also direct determination of the causative agent, and not protein markers, which are the product of his activity, high specificity, high speed is in the case of chlamydia. Direct determination of DNA chlamydia can serve not only as a basis for identifying infection in any form (persistent or active), but also for the analysis of genetic variants of the pathogen.

However, most publications on the development of PCR methods for the detection of chlamydia, built on the detection of only one type of chlamydia.

Thus, the known method of detection of chlamydia species C. trachomatis using PCR using two primers and the matrix in the form of a segment of DNA of chlamydia from receiving amplificate with subsequent determination of the obtained amplification products using horizontal electrophoresis in agarose gel (Griffais R, Thibon M., Detection of Chlamydia trachomatis by the polymerase chain reaction. Environ Res 1989 Feb;140(2): 139-41) [1].

However, this known method is ineffective for clinical use, because in practice, in most cases, the question arises about the complex diagnosis of a wide spectrum of chlamydial infections.

Known method of detection of chlamydial infection by simultaneous detection of all members of the family Chlamydiaceae, including the detection of chlamydia in the material studied by conducting a one-step PCR using two primers and matrix in the requirements. In this known method, the amplification products is determined by the method of horizontal agarose gel electrophoresis (Everett K. D., Andersen, A. A., Identification of nine species of the Chlamydiaceae using PCR-RFLP, Int J Syst Bacteriol 1999 Apr; 49 Pt 2:803-13) [2] is a prototype.

However, this method of detection based on PCR amplification of the gene encoding 16S RNA subunit of the ribosome, is not suitable for use in practical diagnosis, because due to its more conservative gene 16S-RNA compared to the genome OTR PCR system used in this known method, specific not only for members of the family Chlamydiaceae, but also for a number of related chlamydia organisms belonging to the family Simkaniaceae, Parachlamydiaceae and Waddliaceae, as well as for representatives of the genus Mycoplasma.

The technical result achieved by the present group of inventions is a specific detection of chlamydia regardless of the type, combinations of species and presence related to chlamydia species in the sample.

This technical result is achieved in that in the method of diagnosis of chlamydia, including the detection of DNA of chlamydia by conducting a one-step polymerase chain reaction using two primers and the matrix in the form of DNA Klaatu is in use as primers an oligonucleotide probe I:

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and oligonucleotide II:

< / BR>
In doing so, a 32 cycles of PCR, which include one cycle stage of denaturation of 4 min at 95oAnd stage of annealing, 2 min at 60oC, 30 cycles stage of elongation 1 min at 72oWith that stage of denaturation for 40 s at 95oWith stage and annealing 30 s at 60oC and one cycle stage 4 min elongation at 72oC.

This technical result is also achieved by the fact that in the method of diagnosis of chlamydia, including the detection of DNA of chlamydia by conducting a one-step polymerase chain reaction using two primers and the matrix in the form of a segment of DNA of chlamydia from receiving amplificate and subsequent determination of the amplification products, as primers using oligonucleotide II:

< / BR>
and oligonucleotide III:

< / BR>
In doing so, a 32 cycles of PCR, which include one cycle stage of denaturation of 4 min at 95oAnd stage of annealing, 2 min at 60oC, 30 cycles stage of elongation 1 min at 72oWith that stage of denaturation for 40 s at 95oWith stage and annealing 30 s at 60oC and one cycle stage 4 min elongation at 72oC.

This technical result is achieved by the FM definition of amplification products distinctive feature is they spend two-step polymerase chain reaction, and at its first stage we use two external primers in the form of the oligonucleotide I:

< / BR>
and the oligonucleotide II:

< / BR>
and the matrix in the form of a segment of DNA of chlamydia, and the second stage - two internal primers in the form of the oligonucleotide II:

< / BR>
and the oligonucleotide III:

,

as well as the matrix in the form of amplification product obtained in the first stage, and the definition of amplification products is carried out after carrying out the second stage of the polymerase chain reaction.

At both stages are conducted 32 cycles of PCR, which include one cycle stage of denaturation of 4 min at 95oAnd stage of annealing, 2 min at 60oC, 30 cycles stage of elongation 1 min at 72oWith that stage of denaturation for 40 s at 95oWith stage and annealing 30 s at 60oC and one cycle stage 4 min elongation at 72oC.

To create the specific universal PCR detection of chlamydia was conducted computer analysis of the genomes of all known species of the family Chlamydiaceae. The result was identified by the DNA of the gene encoding protein 2 outer membrane (OTR), a length of about 1600 nucleotides, flanked conservative ustavnych plots which flanked identified gene OTR were designed two primers: oligonucleotide I from 21 nucleotides:

< / BR>
and oligonucleotide II of 27 nucleotides:

< / BR>
For conservative area within a fragment in the gene ATR was designed third primer oligonucleotide III of 24 nucleotides:

< / BR>
In addition, for this site as the second primer was used oligonucleotide II of 27 nucleotides, which was designed for one of those conservative areas in which flanked the identified fragment in the gene Omr.

Below are examples illustrating the group of inventions.

Example 1

The working conditions of amplification were performed on the cloned fragments of chlamydial DNA obtained from cell cultures infected with the representatives of the 9 species of the family Chlamydiaceae and kindred the family Simkaniaceae. Preparations of cell cultures obtained from the National Public Health Institute, Finland and the University of California, USA, as well as from the national Institute of Standards and control of medicinal products. Lytvyn, Russia.

When conducting a one-step polymerase chain reaction with Cleotides:

< / BR>
and 9 matrices in the form of DNA representatives of all 9 species of the family Chlamydiaceae, and 3 matrix in the form of DNA representatives of the species Simkania negevensis, Mycoplasma hominis and Mycoplasma pneumoniae with getting amplificate and subsequent determination of the amplification products, in cases when the matrix was used DNA of chlamydia were obtained visible amplicons of approximately 1600 p. O. when using as a template DNA Simkania negevensis, Mycoplasma hominis and Mycoplasma pneumoniae visible amplicons obtained were not.

When this was performed 32 cycles of polymerase chain reaction, which include one cycle stage of denaturation of 4 min at 95oC and stage of annealing, 2 min at 60oC, 30 cycles stage of elongation 1 min at 72oWith that stage of denaturation for 40 s at 95oWith stage and annealing 30 s at 60oC and one cycle stage 4 min elongation at 72oC.

Example 2

Conducted a one-step polymerase chain reaction using as primers the oligonucleotide II of 27 nucleotides:

< / BR>
and the oligonucleotide III of 24 nucleotides:

< / BR>
and 9 matrices in the form of DNA representatives of all 9 species of the family Chlamydiaceae, and 3 matrix in the form of DNA representatives of the species Simkania negevensis, Mycoplasma hominis and Mycoplasma pneumoniae obtaining was allsouls DNA of chlamydia, were obtained visible amplicons of approximately 1030 p. O. when using as a template DNA Simkania negevensis, Mycoplasma hominis and Mycoplasma pneumoniae visible amplicons obtained were not.

When this was performed 32 cycles of polymerase chain reaction, which include one cycle stage of denaturation of 4 min at 95oAnd stage of annealing, 2 min at 60oC, 30 cycles stage of elongation 1 min at 72oWith that stage of denaturation for 40 s at 95oWith stage and annealing 30 s at 60oC and one cycle stage 4 min elongation at 72oC.

Example 3

Conducted a two-stage polymerase chain reaction using at the first stage of the two external primers in the form of I of the oligonucleotide 21 nucleotides:

< / BR>
and the oligonucleotide II of 27 nucleotides:

< / BR>
and 9 matrices form the DNA of the representatives of all 9 species of the family Chlamydiaceae, and 3 matrix in the form of DNA representatives of the species Simkania negevensis, Mycoplasma hominis and Mycoplasma pneumoniae, and at the second stage - two internal primers in the form of the oligonucleotide II of 27 nucleotides:

< / BR>
and the oligonucleotide III of 24 nucleotides:

< / BR>
and 12 matrices in the form of amplification products obtained in the first stage, with the definition of amplification products after performing each step, the polymerase is of 4 min at 95oAnd stage of annealing, 2 min at 60oC, 20 cycles stage of elongation 1 min at 72oWith that stage of denaturation for 40 s at 95oWith stage and annealing 30 s at 60oC and one cycle stage 4 min elongation at 72oWith, and at the second stage - 27 cycles of PCR, which include one cycle stage of denaturation of 4 min at 95oAnd stage of annealing, 2 min at 60oC, 25 cycles stage of elongation 1 min at 72oWith that stage of denaturation for 40 s at 95oWith stage and annealing 30 s at 60oC and one cycle stage 4 min elongation at 72oC. Despite the fact that at 22 cycles of the first stage PCR failed to get visible amplicons, when used as a matrix obtained amplificata in subsequent PCR (second stage) in cases when the matrix was used DNA of chlamydia were obtained visible amplicons of approximately 1030 p. O. In case of use as a matrix in the first stage, DNA Simkania negevensis, Mycoplasma hominis and Mycoplasma pneumoniae visible amplicons neither in the first nor in the second stage have not been received.

Thus, the results suggest that the proposed methods for the detection of chlamydia is simple in execution, cheaper separate test systems and high specificity. Offers the AI PCR. The presence of the representatives of the other, even closely related to chlamydia species does not interfere with the determination.

1. A method for the diagnosis of chlamydial infection, including the detection of DNA of chlamydia in the material studied by conducting a one-step polymerase chain reaction using two primers and the matrix in the form of a segment of DNA of chlamydia from receiving amplificate and subsequent determination of the obtained amplification products, wherein in use as primers an oligonucleotide probe I:

< / BR>
and oligonucleotide II:

< / BR>
2. A method for the diagnosis of chlamydial infection by p. 1, characterized in that hold 32 rounds of polymerase chain reaction, which include one cycle stage denaturate 4 min at 95oAnd stage of annealing, 2 min at 60oC, 30 cycles stage of elongation 1 min at 72oWith that stage of denaturation for 40 s at 95oWith stage and annealing 30 s at 60oC and one cycle stage 4 min elongation at 72oC.

3. A method for the diagnosis of chlamydial infection, including the detection of DNA of chlamydia in the material studied by conducting a one-step polymerase chain reaction using two primers and the matrix in the form of a segment of DNA of chlamydia from receiving amplificata sportsouth oligonucleotide II

< / BR>
and oligonucleotide III:

< / BR>
4. A method for the diagnosis of chlamydial infection on p. 3, characterized in that hold 32 rounds of polymerase chain reaction, which include one cycle stage of denaturation of 4 min at 95oAnd stage of annealing, 2 min at 60oC, 30 cycles stage of elongation 1 min at 72oWith that stage of denaturation for 40 s at 95oWith stage and annealing 30 s at 60oC and one cycle stage 4 min elongation at 72oC.

5. A method for the diagnosis of chlamydial infection, including the detection of DNA of chlamydia from receiving amplificate and subsequent determination of the obtained amplification products, wherein spend two-step polymerase chain reaction, and at its first stage we use two external primers in the form of the oligonucleotide I

< / BR>
and the oligonucleotide II

< / BR>
and the matrix in the form of a segment of DNA of chlamydia, and in the second stage, two internal primers in the form of the oligonucleotide II

< / BR>
and the oligonucleotide III

< / BR>
and the matrix in the form of amplification product obtained in the first stage, and the definition of amplification products is carried out after carrying out the second stage polymerase chain reaction.

6. A method for the diagnosis of chlamydial infection on p. 5, on the stage of denaturation of 4 min at 95oAnd stage of annealing, 2 min at 60oC, 30 cycles stage of elongation 1 min at 72oWith that stage of denaturation for 40 s at 95oWith stage and annealing 30 s at 60oC and one cycle stage 4 min elongation at 72oC.

 

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