RU2385945C1 - Method for prediction of human or simian manifested or obliterated chlamidia infection and kit for implementation thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: DNA is recovered from a clinical material or cell culture infected with Chlamydia recovered from the clinical material. It is followed with multiplex PCR with primers Ctr 5'-TGGCGATATTTGGGCATCC-3, R 5'-CTTCTTTACCTGGTACGCTC-3, PLf 5'-TCCGGAGCGAGTTACGAAGA-3' and PLr 5' -AATCAATGCCCGGGATTGGT-3'. Then amplification products are exposed to electrophoretic analysis to predict: manifested human or simian Chlamydia infection in case of positive PCR with primers Ctr 5'-TGGCGATATTTGGGC ATCC-3, R 5' -CTTCTTTACCTGGTACGCTC-3', PLf 5'-TCCGGAGCGAGTTACGAAGA-3' and PLr 5'-AATCAATGCCCGGGATTGGT-3'; obliterated human or simian Chlamydia infection in case of positive PCR with primers Ctr 5'-TGGCGATATTTGGGCATCC-3', R 5'-CTTCTTTACCTGGTACGCTC-3' and negative PCR with primers PLf 5' -TCCGGAGCGAGTTACGAAGA-3' and PLr 5' -AATCAATGCCCGGGATTGGT-3'.

EFFECT: invention can be used to diagnose human or simian Chlamydia infection, to predict both manifested and obliterated human and simian Chlamydia infection caused by Chlamydia trachomatis with simplifying the diagnostic technique for Chlamydia infection.

2 cl, 3 ex

Description

The invention relates to medicine and biology and can be used to diagnose chlamydial infections in humans and monkeys.

Chlamydia is a widespread group of intracellular obligate parasites that cause diseases in humans and animals. Among the causative agents of chlamydia, Chlamydia trachomatis, which causes various infectious diseases, including infections of the urogenital tract, trachoma, venereal lymphogranuloma, inflammatory diseases of the lymphadenoid pharyngeal ring, infectious pathology of the respiratory system and conjunctiva of the newborn, is of particular importance for medicine at present.

For an informative study of the clinic, pathogenesis and treatment of chlamydial infections, it is necessary to use the optimal experimental model. Such a model can be monkeys as animals closer to humans along the evolutionary ladder. Experimental infection of these animals with chlamydia isolated from humans is known (Patton DL, Cosgrove YT, Kuo Cho-Chou, Campbell LA Effects of quinolone analog CI-960 in a monkey model of Chlamydia trachomatis salpingitis. // Antimicrobial agents and chemotherapy. - 1993. - Vol. 37, N 1. - P.8-13). However, prior to our studies, it was not known that monkeys had their own chlamydial infection. In this regard, in order to eliminate the false results of an experimental study of chlamydial infection caused by Chlamydia trachomatis, we conducted studies to identify species of monkeys that are promising for use as a model for studying chlamydia, their own chlamydial infection caused by this type of chlamydia. As a result of our studies, it was found that monkeys have their own diseases of the urogenital tract, respiratory and visual organs caused by Chlamydia trachomatis. It is known that in humans infections caused by Chlamydia trachomatis can occur in the form of manifest or erased forms, and this requires the earliest possible selection of the optimal treatment options (Interferon status, interferon drugs in the treatment and prevention of infectious diseases and rehabilitation of patients). / Edited by S.S. Afanasyev, G.G. Onishchenko, V.A. Aleshkin, L.V. Feklisova, M.S. Afanasyev, A.V. Aleshkina. - M .: Triada-X. - 2005. - S. 421-465; RU patent No. 2222018, 2004.01.20, G01N 33/53). During dynamic observations, we found that in monkeys, fresh and chronic infections caused by Chlamydia trachomatis, as well as in humans, can occur in the form of predominantly manifest or predominantly erased forms (the term "predominant form" is used to denote the form of infection that is more often observed during a dynamic examination of a particular animal).

Therefore, we developed a method based on the most sensitive method for the detection of chlamydia - polymerase chain reaction (PCR), which provides chlamydia caused by Chlamydia trachomatis, both in humans and in monkeys, an early choice of the optimal treatment options for the forms of chlamydial infection due to the possibility of differentiated forecasting as manifest and erased forms of chlamydial infection caused by Chlamydia trachomatis.

A known method for the diagnosis of chronic urogenital chlamydia by assessing resistance to chlamydial infection, which further determines the expression levels of TL receptor-2 and TL receptor-4 in real-time PCR using urethral scraping material and diagnoses chronic urogenital chlamydia when the expression levels in the scraping material from the urethra of TL-receptor-2 are not more than 5 units and TL-receptor-4 is not more than 5 units (RU patent No. 23237995, 2008.06.27, G01N 33/569, C12Q 1/68).

The disadvantage of this method is that it cannot be used for differentiated prediction of manifest and erased forms of chlamydial infection in both humans and monkeys, due to the lack of determination of combinations of nucleotide sequences of Chlamydia trachomatis, typical for pathogen isolates that cause manifest or erased forms of chlamydial infection.

It is known that the expression of 16S ribosomal RNA is usually associated with the clinically active course of chlamydia in a patient with trachoma. Detection of the omp1 DNA gene is less frequently associated with the active course of this chlamydia (Burton MJ, Holland MJ, Jeffries D., Mabey DCW, Bailey RL Conjunctival chlamydial 16S ribosomal RNA expression in trachoma: is chlamydial metabolic activity required for disease to develop? // Clinical Infectious Diseases. - 2006. - Vol.42, N 4. - P.463-470).

However, according to our data, 16S ribosomal RNA genes are detected in most cases of clinical manifestations of chlamydial infection in humans and monkeys without regard to the form of the disease, which does not allow their identification to be used for differentially predicting manifest or erased forms of human chlamydial infection or monkeys caused by Chlamydia trachomatis.

A known method for the diagnosis of manifest and latent forms of chronic urogenital chlamydia in men by determining the DNA of Chlamydia trachomatis, specific immunoglobulins (G, A) to Chlamydia trachomatis and assessing foci of a chronic infection process, which in the presence of negative confirmatory tests for chlamydia in men (PCR and IgA) complemented by the determination of Chlamydia trachomatis DNA, specific immunoglobulins (G, A) to Chlamydia trachomatis and foci of a chronic infection process in a woman with constant sexual intercourse (at least 3 months eggs) without barrier methods of protection, and with a positive PCR test and / or the presence of class A immunoglobulins to Chlamydia trachomatis in the blood, as well as in the presence or absence of one or more chronic foci, a woman manifests a manifest or latent form of chronic urogenital chlamydia, in a man in this case and subject to the presence of one or more chronic foci of infection and a diagnostic titer of immunoglobulins G, a manifest form of urogenital chlamydia is diagnosed, and in the absence of clinical and Athorne signs of infection diagnosed latent form (RU patent №2222018, 2004.01.20, G01N 33/53).

The disadvantage of this method is that it cannot be used for differentially predicting the manifest and erased forms of chlamydial infection in both humans and monkeys, due to the lack of determination of combinations of nucleotide sequences of Chlamydia trachomatis, typical for pathogen isolates that cause manifest or erased forms of chlamydial infection. In addition, it is difficult due to the large list of used options for laboratory methods.

A known method for the diagnosis of persistent or chronic infection in a patient caused by a microorganism of the Chlamydiaceae family, comprising detecting in the biological sample obtained from the patient, relative to the lytic phase of the chlamydia development cycle, changing the level and / or functional activity of the gene expression product selected from ruk, nlpd, CpnO585 or a gene belonging to the same regulatory or biosynthetic pathway as ruk, nlpd, CpnO585, ompA, oynpB, hsp60, or as a gene involved in LPS biosynthesis, or a variant of the above gene.

According to a known method for analysis of gene expression, real-time PCR or PCR is carried out as an option using primers specific for Chlamydophila pneumoniae:

Ct16S-F2 5'-GGATTTATTGGGCGTAAAGG

Ct16S-R 5'-TCCACATCAAGTATGCATCG

CpnompA-F 5'-GCTGCAAACTATACTACTGC

CpnompA-R 5'-GAAAACATCAAAGCGATCCC

CpnompB-F 5'-GTGATGGGAAATTAGTCTGG

CPNOMPB-R 5'-ATCCTGTGTTCACTACTTCG

CpnomcB-F 5'-AGCAGAAGTTTACTCTGTCG

CpnomcB-R 5'-CTACTGATGGAAACCTAAGC

Cpn76kDa-F 5'-AAGATATCAAGGCTACTGATGAGGAAACCG

Cpn76kDa-R 5'-TTGATATCTAGAACTTGCTGCAGCGGGA

CPNPMP1-F 5'-GACTACTGCTATAGGTAAGG

Cpnpmp1-R 5'-GAGATGCTAAGTTTCCTAGC

Cpng1tX-F 5'-TCTCTTTCGTCCATTGATCG

Cpng1tX-R 5'-CTCAGGATTGTTAGAGTACC

CPNHSP60B-F 5'-GTCCAGTGAAATCATGGCCG

Cpnhsp60AI-5'-CCCATGTTTTCATGTTTGTC

CpnyaeT-F 5'-TCAGGAAATCAAGTCGTTCC

CpnyaeT-R 5'-AGATTCCTGAGAACGTAAGC

Cpnpyk-F 5'-TGTTGTTGTCTCTTCAGAGG

CPNPYK-R 5'-CTACCCCAAACTTAAGATCC

CPNN1PD-F 5'-TCAATGATCTTACCACCACC

Cpnn1pD-R 5'-GTTACGCAATGCTATTGTCC

CPN0585-F 5'-TGCATCTTATATAGAGAGCTCG

CPN0585-R 5'-GAAGTTAGCGGATTTAGAGG

CPN1046-F 5'-GAGGAGAACTGATAAGAACG

Cpn1046-R 5'-CTTAACTCCTGATCTCATCC.

To measure the concentration of the polypeptide, the product of the expression of the above genes, the biological sample is mixed with antigen-binding molecules that specifically interact with this polypeptide, the concentration of the complex including the polypeptide and antigen-binding molecule is measured, and the concentration of the polypeptide in the test sample is determined by the concentration of the complex. To determine the level of the product of expression of the above genes, the biological sample can also be mixed with an antigen corresponding to at least a portion of the polypeptide encoded by the gene. In this case, determine the level of antigen-specific proliferation of T-lymphocytes. Diagnostically significant is a 10% change in the level and / or functional activity of the gene expression product (WO patent No. 02/14516, 2002.02.21, C12N 15/30).

The main disadvantages of the known method for the diagnosis of persistent or chronic infection are the following:

- the known method cannot be used for differentially predicting the manifest and erased forms of chlamydial infection in both humans and monkeys, due to the lack of determination of combinations of nucleotide sequences of Chlamydia trachomatis, typical of pathogen isolates that cause manifest or erased forms of chlamydial infection;

- the known method is complicated due to the large list of used options for laboratory methods;

- the sequence of primers used in the known method and their kinetics does not allow for multiplex detection of the expression of different genes, which further complicates the method in connection with separate PCR to detect the expression of each gene.

The basis of the invention is the task of expanding the possibility of using PCR to predict both manifest and erased forms of chlamydial infection in humans and in monkeys caused by Chlamydia trachomatis, while simplifying the method of diagnosing chlamydial infection.

The problem is solved in that in order to predict a manifest or erased form of chlamydial infection in humans or monkeys caused by Chlamydia trachomatis, after DNA is isolated from clinical material or a cell culture infected with chlamydia isolated from clinical material, multiplex PCR with primers Ctr 5'-TGGCGATATTTGGCG 3 ', R 5'-CTTCTTTACCTGGTACGCTC-3', PLf 5'-TCCGGAGCGAGTTACGAAGA-3 'and PLr 5'-AATCAATGCCCGGGATTGGT-3' and then electrophoretic analysis of amplification products and predict: a manifest form of a chlamydial infection with a chlamydial infection imeters Ctr 5'-TGGCGATATTTGGGCATCC-3 ', R 5'-CTTCTTTACCTGGTACGCTC-3', PLf 5'-TCCGGAGCGAGTTACGAAGA-3 'and PLr 5'-AATCAATGCCCGGGATTGGT-3; erased form of chlamydial infection of humans or monkeys with positive PCR with primers Ctr 5'-TGGCGATATTTGGGCATCC-3 ', R 5'-CTTCTTTACCTGGTACGCTC-3' and negative PCR with primers PLf 5'-TCCGGAGCAGGGT 3-A 3 PLG A '. The kit for the implementation of the proposed method contains primers Ctr 5'-TGGCGATATTTGGGCATCC-3 ', R 5'-CTTCTTTACCTGGTACGCTC-3', PLf 5'-TCCGGAGCGAGTTACGAAGA-3 'and PLr 5'-AATCAATGCCCGG enzyme-linked 3' tag, enzyme tag for the reaction, a mixture of four deoxynucleotide triphosphates, positive control, negative control, wax and oil for PCR.

Our studies have allowed us to develop a combination of two new pairs of primers (Ctr and R, PLf and PLr) specific for Chlamydia trachomatis, suitable for use in multiplex PCR, expanding the possibility of using PCR for differentiated prediction of both manifest and erased forms of chlamydial infection and in humans and in monkeys caused by Chlamydia trachomatis, while simplifying the method for diagnosing chlamydial infection.

The inventive method for predicting the form of chlamydial infection is new and is not described in the literature.

The technical result of the claimed invention is the provision of expanding the possibility of using PCR to predict both manifest and erased forms of chlamydial infection in both humans and monkeys caused by Chlamydia trachomatis, while simplifying the method of diagnosing chlamydial infection by combining two new primer pairs (Ctr and R , PLf and PLr) specific for Chlamydia trachomatis, suitable for use in multiplex PCR.

The invention is illustrated by the following examples, when using the proposed method for predicting the form of chlamydial infection and the claimed kit for its implementation, the provision of prediction of both manifest and erased forms of chlamydial infection in humans and in monkeys caused by Chlamydia trachomatis, while simplifying the method of diagnosing chlamydia infection.

Example 1. 19 monkeys of the rhesus macaque species and Javanese macaque species of both sexes, aged 5 to 10 years, with inflammatory diseases of the urogenital tract.

Initially, DNA was isolated from the scraping material of the mucous membrane of the cervical canal of the cervix, animal urethra. Then, multiplex PCR was performed using a kit for predicting a manifest or erased form of human or monkey chlamydial infection caused by Chlamydia trachomatis containing primers: Ctr 5'-TGGCGATATTTGGGCATCC-3 ', R 5'-CTTCTTTACCTGGTACGGGTC-3-CTCTAG-5 3 'and PLr 5'-AATCAATGCCCGGGATTGGT-3', thermostable Tag polymerase enzyme, reaction buffer, a mixture of four deoxynucleotide triphosphates, positive control, negative control, wax and PCR oil, followed by electrophoretic analysis of amplification products.

As a result of the study, positive monkey PCR with primers Ctr 5'-TGGCGATATTTTGGGCATCC-3 ', R 5'-CTTCTTTACCTGGTACGCTC-3', PLf 5'-TCCGGAGCGAGTTACGAAGA-3 'and PLr 5'GTGGATGACC-3' and was established in 8 monkeys. Based on the results of PCR in these monkeys, a manifest form of chlamydial infection caused by Chlamydia trachomatis was predicted, which was confirmed by their subsequent clinical and laboratory examinations.

In the remaining 11 monkeys, positive PCR was established with primers Ctr 5'-TGGCGATATTTGGGCATCC-3 ', R 5'-CTTCTTTACCTGGTACGCTC-3'. Based on the results of PCR in these monkeys, the erased form of chlamydial infection caused by Chlamydia trachomatis was predicted, which was confirmed by their subsequent clinical and laboratory examinations.

Example 2. Patient S., age 19 years. The clinical diagnosis is urethritis.

Initially, DNA was isolated from a cell culture infected with scraping material from the patient's urethral mucosa. Then, multiplex PCR was performed using a kit for predicting a manifest or erased form of human or monkey chlamydial infection caused by Chlamydia trachomatis containing primers: Ctr 5'-TGGCGATATTTGGGCATCC-3 ', R 5'-CTTCTTTACCTGGTACGGGTC-3-CTCTAG-5 3 'and PLr 5'-AATCAATGCCCGGGATTGGT-3', thermostable Tag polymerase enzyme, reaction buffer, a mixture of four deoxynucleotide triphosphates, positive control, negative control, wax and PCR oil, followed by electrophoretic analysis of amplification products.

As a result of the study, positive PCR with primers Ctr 5′-TGGCGATATTTGGGCATCC-3 ′, R 5′-CTTCTTTACCTGGTACGCTC-3 ′, PLf 5′-TCCGGAGCGAGTTACGAAGA-3 ′ and PLr 5′-AATGATGATG was established. Based on the results of PCR in the examined patient, a manifest form of chlamydial infection caused by Chlamydia trachomatis was predicted, which was confirmed by subsequent clinical and laboratory examinations.

Example 3. Patient A., age 28 years. I turned to the diagnostic center with complaints of the absence of pregnancy. The clinical diagnosis is infertility.

Initially, DNA was isolated from the scraping material of the mucous membrane of the cervical canal of the cervix of the patient. Then, multiplex PCR was performed using a kit for predicting a manifest or erased form of human or monkey chlamydial infection caused by Chlamydia trachomatis containing primers: Ctr 5'-TGGCGATATTTGGGCATCC-3 ', R 5'-CTTCTTTACCTGGTACGGGTC-3-CTCTAG-5 3 'and PLr 5'-AATCAATGCCCGGGATTGGT-3', thermostable Tag polymerase enzyme, reaction buffer, a mixture of four deoxynucleotide triphosphates, positive control, negative control, wax and PCR oil, followed by electrophoretic analysis of amplification products.

As a result of the study, positive PCR with primers Ctr 5'-TGGCGATATTTGGGCATCC-3 ', R 5'-CTTCTTTACCTGGTACGCTC-3' was established. Based on the results of PCR in the examined patient, the erased form of chlamydial infection caused by Chlamydia trachomatis was predicted, which was confirmed by subsequent clinical and laboratory examinations.

Thus, the above examples prove the applicability of the proposed method for predicting both the manifest and erased forms of chlamydial infection in humans and in monkeys caused by Chlamydia trachomatis using only two pairs of primers, which expands the possibility of using PCR to predict the form of chlamydial infection while simplifying the method.

Claims (2)

1. A method for predicting a manifest or erased form of chlamydial infection in humans or monkeys caused by Chlamydia trachomatis, characterized in that after DNA is isolated from a clinical material or a cell culture infected with chlamydia isolated from clinical material, multiplex PCR is carried out with Ctr 5'-TGGCGATATTTGGCC primers -3 ', R 5'-CTTCTTTACCTGGTACGCTC-3', PLf 5'-TCCGGAGCGAGTTACGAAGA-3 'and PLr 5'-AATCAATGCCCGGGATTGGT-3' and then electrophoretic analysis of amplification products and predict:
the manifest form of chlamydial infection of humans or monkeys with positive PCR with primers Ctr 5'-TGGCGATATTTGGGCATCC-3 ', R 5'-CTTCTTTACCTGGTACGCTC-3', PLf 5'-TCCGGAGCGAGTTACGAAGA-3'GGATGAT-3GT-3GT-3GTCA erased form of chlamydial infection of humans or monkeys with positive PNR with primers Ctr 5'-TGGCGATATTTGGGCATCC-31, R 5'-CTTCTTTACCTGGTACGCTC-3 'and negative PCR with primers PLf 5'-TCCGGAGCGAGGTG 3-GATT 3 PLTA . 2. A kit for predicting the manifest or erased form of chlamydial infection of humans or monkeys caused by Chlamydia trachomatis, characterized in that it contains primers Ctr 5'-TGGCGAT ATTTGGGC ATCC-3 ', R 5'-CTTCTTTACCTGGTACGCTC-3', TCCGGAGCGAGTTACGAAGA-3 'and PLr 5'-AATCAATGCCCGGGATTGGT-3', thermostable Tag polymerase enzyme, reaction buffer, a mixture of four deoxynucleotide triphosphates, positive control, negative control, wax and PCR oil.