RU2533238C1 - Method for mycoplasma hominis dna detection from blood samples - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention represents a method for the Mycoplasma hominis DNA detection from a blood sample by dividing it into a supernatant and precipitate by centrifugation followed by enriching the analysed material, recovering DNA by affine sorption with a sorbent and a lysis buffer added, incubating, washing the sorbent precipitate three times by centrifugation and removing the supernatant, drying the sorbent precipitate, adding a DNA elution solution and detecting by the PCR method. The method is characterised by the fact that the material to be analysed is a whole blood precipitate and to be enriched while recovering DNA by doubling up a number of test tubes per one sample with the whole blood precipitate 100 mcl and the sorbent 20 mcl in each; before the washing procedure, each test tube is added with the washing solution 375 mcl, while the content of the test tubes is combined in one test tube with adding the DNA elution solution 40 mcl to the sorbent precipitate.

EFFECT: higher diagnostic accuracy.

1 tbl

Description

The invention relates to medicine, namely to methods for detecting molecular markers of Mycoplasma hominis (M. hominis) for further use in the diagnostic algorithm.

M. hominis is classified as opportunistic microorganisms, the prevalence of which in various population groups varies from 10 to 50% (according to several authors, up to 80%). M. hominis can normally colonize the organs of the urogenital system and are often detected in clinically healthy individuals (5-20%). However, under certain conditions, these microorganisms can cause the development of inflammatory processes of the genitourinary tract [1].

Infection occurs mainly through sexual contact. There is also intrauterine infection of the fetus and infection of the newborn when passing through the infected birth canal of the mother. M. hominis can cause non-gonococcal urethritis and prostatitis, pelvic inflammatory diseases (salpingitis, oophoritis, endometritis, adnexitis), pregnancy and fetal pathology, infertility in women and men, as well as perinatal infections of newborns. The social significance of urogenital mycoplasma infection consists in its complications: violation of reproductive function in the form of infertility, threatened abortion, frequent prematurity, placenta pathology, fetal infection [2].

Examination for urogenital mycoplasmas is carried out according to clinical indications. The main methods for identifying mycoplasmas are:

1. Molecular biological methods based on the amplification of specific DNA / RNA fragments from the urogenital tract of difficult to cultivate pathogens M. hominis, using test systems approved for medical use in the Russian Federation.

2. Cultural (bacteriological) research for identification of M. hominis. Mycoplasmas are very demanding on nutrient media and are hardly distinguished by the cultural method from patients, especially in chronic infections.

The method of polymerase chain reaction (PCR) allows you to identify the genetic material of the pathogen, has high sensitivity and specificity. The result of the analysis does not depend on the state of the immune system and stage of the disease, but is determined only by the fact of the presence of the pathogen, or its genetic material in the test sample. The study allows the detection of various types of mycoplasmas. The test is used to confirm the diagnosis of generalized forms of mycoplasma infection [3].

The basis for DNA isolation by affinity sorption is selective sorption on the surface of the silica gel in the presence of a chaotropic salt of lysis buffer. Inhibitors and other components of the test material, which can lead to a worsening of the reaction, remain in solution. Using centrifugation, silica gel with adsorbed DNA is precipitated, and the supernatant with PCR inhibitors is removed. A series of subsequent washes provides highly purified DNA, so this technique is universal and suitable for a wide range of biological samples, including samples with a high protein content, such as biopsy specimens or whole blood, as well as for detecting various infections with maximum sensitivity. In the diagnostic study of blood serum samples of patients, false-negative results are possible, because studies have shown that in serum mycoplasmas are detected much less frequently than in the urogenital tract [4].

To obtain serum, blood tubes are assayed for 30 minutes until a clot forms completely. Then centrifuged at 3000 rpm for 10 minutes at room temperature [5]. Preliminary preparation of blood samples to obtain serum (supernatant) promotes precipitation together with blood cells in the sediment and life forms of the desired microorganisms, which are not used in diagnostics, which leads to a decrease in the value of serum as an object of study for the diagnosis of M. hominis.

Therefore, the diagnosis of mycoplasma infection cannot be based on the detection of DNA in blood serum by PCR. At the same time, this method may be useful for studying the ways and means of spreading mycoplasmas in the body, generalization of infection, and pathogenesis mechanisms [2]. So, in the blood serum of patients with chronic urogenital mycoplasma infection, with chronic fatigue syndrome and a number of other chronic pathological conditions, it was possible to detect mycoplasma DNA and ureaplasma by PCR with negative results of the culture method [6].

Detection of M. hominis DNA from the blood is highly informative for establishing the causes of chronic, low-symptomatic inflammatory diseases of the upper and lower parts of the urogenital tract, joint damage, which is an indicator of the generalization of infection. However, the low content of the infectious agent in blood samples requires certain approaches to increase the information content of the method.

The closest solution is the detection of human urogenital mycoplasmas (M. hominis) in blood serum (supernatant) by PCR [7]. A 1 ml aliquot of blood serum was centrifuged (pre-enrichment). 100 μl of transport medium was added to the precipitate, and DNA was isolated by affinity sorption (according to the instructions [8]).

Amplification of DNA was carried out in a Tertsik amplifier, and its detection was performed using agarose gel electrophoretic analysis.

In this way, in the blood serum of M. hominis was detected in 8% of cases.

The objective of the invention is to improve the method for detecting M. hominis DNA from blood.

The technical result that will be achieved by using the invention is to improve the accuracy of the diagnosis of M. hominis.

The technical result is achieved by the fact that in the method of isolating M. hominis DNA from blood samples by separating it by centrifugation into a supernatant and a precipitate, subsequent enrichment of the test material, DNA isolation by affinity sorption with the addition of a sorbent and lysis buffer, incubation, three times washing of the sorbent precipitate by centrifugation and removal of the supernatant, drying of the sorbent precipitate, addition of a solution for DNA elution and detection by PCR method; blood enrichment, and its enrichment is carried out in the process of DNA extraction by doubling the number of tubes per sample with 100 μl of whole blood sediment and 20 μl of sorbent, to which, before the third washing, 375 μl of the washing solution are added, and their contents are combined into one tube and 40 μl of DNA elution solution was added to the sorbent precipitate.

The invention consists in certain approaches that enhance the accuracy of diagnosis (detection) of M. hominis DNA in the blood:

1. The choice as the object of study - the whole blood sediment containing the largest number of bacterial particles.

2. Considering the biological feature - the content of the infectious agent in the blood in quantities on the verge of detection, an additional stage of enrichment of the sample (test material) in the process of DNA extraction is included, which allows you to analyze twice the amount of whole blood sediment without reducing the efficiency of DNA sorption on silica gel and without increasing the content undesirable impurities that inhibit PCR, while:

- lysis of the sample in the presence of silicogel in two separate tubes creates spatially optimal conditions for the passage of the lysis process and promotes effective sorption of DNA and washing from inhibitors;

- 20 μl of a sorbent solution is a necessary and sufficient amount (experimentally selected) for complete sorption of DNA obtained from 100 μl of lysed whole blood

- the use of a washing solution in an amount of 375 μl is selected to half-fill the eppendorf tube (max. volume 1,500 μl) when the sorbent solution is combined from two tubes into one and does not reduce the quality of the third sorbent washing;

- the addition of a sorbent to each tube of 20 μl (total volume of 40 μl) and the output of sorbed DNA in 40 μl of an elution solution increases the concentration of DNA and the likelihood of the target DNA getting into the amplification reaction with specific primers.

From the analysis of scientific, technical and patent literature, the use of whole blood sediment as a test sample and its enrichment before M. hominis DNA is isolated by PCR by the claimed combination of techniques provides an increase in the probability of the DNA of the desired agent entering the sample, which increases the chances of producing specific DNA during amplification - those. the chances of a positive result in the presence of an infectious agent, i.e. a significant increase in the accuracy of diagnosis, we have not identified, which allows us to conclude that the proposed solutions meet the criteria of "novelty" and "inventive step".

The invention is as follows.

Whole venous blood taken in a tube with an anticoagulant is centrifuged at 3000 rpm for 20 minutes at room temperature. Then the whole plasma (supernatant) is taken, and the precipitate is taken for examination by PCR.

20 μl of sorbent and 100 μl of whole blood sediment are added to all tubes, vortexed and incubated at room temperature for 7 minutes, constantly stirring. Then the sorbent is precipitated by centrifugation at 5 thousand rpm for 30 seconds. Remove the supernatant using a vacuum aspirator and a separate tip without filter for each pair of tubes.

Then carry out washing No. 1. Why add 300 μl of solution for washing No. 1, mix on a vortex until complete resuspension of the sorbent. The sorbent is precipitated by centrifugation at 5 thousand rpm for 30 seconds. Remove the supernatant using a vacuum aspirator and a separate tip without filter for each pair of tubes. Washing No. 2 is carried out with 500 μl of washing solution No. 2 added to the samples, mixed with a vortex until the sorbent is fully resuspended. The sorbent is precipitated by centrifugation at 5 thousand rpm for 30 seconds. Remove the supernatant using a vacuum aspirator and a separate tip without filter for each pair of tubes. Washing No. 3 is carried out with the addition of 375 μl of washing solution No. 2 to the samples, mixed with a vortex until the sorbent is fully resuspended. Then the suspension of the sorbent from one tube is transferred by a 1000 μl dispenser with a disposable tip and a filter to the second sample tube, thereby combining into one sample containing 700 μl of the suspension. Vortexing. The sorbent is precipitated by centrifugation at 5 thousand rpm for 30 seconds. Remove the supernatant using a vacuum aspirator and a separate tip for each sample. A tube with an open cap containing a sorbent precipitate is placed in a thermostat at T 65 ° C for 10-15 minutes to dry it.

Then, the DNA elution step is carried out: 40 μl of the DNA elution solution is added, mixed on a vortex and placed in a thermostat at 65 ° C for 5 minutes, periodically shaking on a vortex. Centrifuge the tube at 12 thousand rpm for 1 minute in a microcentrifuge. Discard the supernatant in a clean tube. DNA is ready for PCR. To isolate M. hominis DNA from whole blood, any silica gel kits designed to isolate DNA from whole blood can be used as a lysing, washing solution, solution for elution of DNA.

A comparison of the detection efficiency of M. hominis DNA by the claimed method and the prototype method is presented in the table.

As can be seen from the table, the claimed method is more informative: it provides an average of 35 times greater yield of specific DNA compared to the prototype, and therefore increases the chances of a positive result in the presence of an infectious agent, and therefore increases the release of M. hominis DNA from the precipitate whole blood 1.2 times compared with excretion from the urogenital tract. Thus, the inventive method can be used to detect DNA of M. hominis in whole blood sediment using PCR to diagnose urogenital mycoplasmas.

Literature

1. Management of patients with sexually transmitted infections and urogenital infections. Clinical recommendations. Russian Society of Dermatovenerologists and Cosmetologists. M. 2012. Business Express. S. 74-78.

2. Tapilskaya N.I. The use of Vilprafen in patients with infertility and habitual miscarriage at the stage of pregravid preparation / Difficult patient. - 2010. No. 1-2. S.13-18.

3. Zigangirova N.A. Persistence of pathogenic mycoplasmas and its molecular genetic mechanisms / Abstract of Diss. ... doctor. biol. Sciences, 2001.46 p.

4. Balabanov D.N. Antigenemia with urogenital mycoplasma infections / Abstract. diss. ... cand. honey. Sciences, Moscow, 2009. - 31 p.

5. Methodical recommendations. Fence, transportation, storage of clinical material for PCR diagnostics. Central Research Institute of Health of the Russian Federation, Moscow, 2003. P. 4.

6. Rakovskaya I.V. et al. Duration of preservation of viable cells, DNA and antigens of Mycoplasma hominis and ureaplasmas in human serum at 37 ° C "Clin. Laboratory diagnostics", 2008, No. 11, P.40-42.

7. Barkhatova O.I. et al. "Detection of human urogenital mycoplasmas in blood serum" "Clin. Lab. Diagnostics", 2008, No. 3, S.49-51.

8. Instructions for the use of a complex of reagents for the isolation of DNA from the clinical material “DNA-sorb-B”, approved by Order of the Federal Service for Health and Safety of 13.07.09 No. 5535-Pr / 09. C.3-4.

Claims (1)

The method of isolation of Mycoplasma hominis DNA from blood by separation by centrifugation on a supernatant and a precipitate, subsequent enrichment of the test material, DNA isolation by affinity sorption with the addition of a sorbent and lysis buffer, incubation, three-time washing of the sorbent precipitate by centrifugation and removal of the supernatant, drying the sorbent a solution for elution of DNA and detection by PCR method, characterized in that the whole blood sediment is used as the test material, and its enrichment is They are produced in the process of DNA extraction by doubling the number of tubes per sample with 100 μl of a whole blood precipitate and 20 μl of sorbent in each, to which 375 μl of a washing solution is added before the third washing, and their contents are combined into one tube and a solution is added to the sorbent precipitate for elution of DNA in an amount of 40 μl.