RU2231790C2 - Method for treatment of whole blood for isolating tuberculosis mycobacterium dna by polymerase chain reaction - Google Patents

Abstract

FIELD: medicine, biochemistry.

SUBSTANCE: invention relates to a method for preparing blood sample for isolating Mycobacterium tuberculosis DNA by using polymerase chain reaction. Method involves lysis of erythrocytes and washing out a precipitate from hemoglobin followed by using the washed out precipitate for isolation of DNA and its assay by method of polymerase chain reaction. For lysis of erythrocytes the solution NaOH/NaCl is used as early but in the ration allowing to avoid formation of clots and blood coagulation, namely, blood : 2% NaOH/0.125% NaCl = 5:1. Hemoglobin from a precipitate is removed with a buffer 10 mM tris-HCl and 1 mM EDTA (pH 7.6) with 1% of Triton. The advantage of invention involves accelerating the sample preparing and reducing blood sample amount from a patient without reducing sensitivity in carrying out the polymerase chain reaction.

EFFECT: improved treatment method.

Description

The invention relates to medicine, in particular to a method for treating whole blood to detect M. tuberculosis DNA (MBT) by polymerase chain reaction (PCR).

The increase in the incidence of tuberculosis and the direct dependence of the effectiveness of treatment of patients on the timing of its detection make the task of early and specific diagnosis of this disease one of the most pressing problems of modern phthisiology.

In recent years, for these purposes, along with generally accepted microbiological methods in phthisiology, the modern molecular genetic method, the polymerase chain reaction, has been widely used, which has established itself as the fastest, most sensitive and specific test for detecting MBT DNA. The method is based on the multiple copying of a specific MBT DNA fragment using specially selected primers and the DNA polymerase enzyme. The material for detecting MBT DNA by PCR is various biological secrets obtained from patients with pulmonary and extrapulmonary pathology.

Of particular interest for the detection of MBT DNA is peripheral blood, as the most real secret for screening the population for the mass detection of cases of tuberculosis. The most important factor affecting the detection of MBT DNA using the PCR method in the blood is the method of processing the primary material, especially the release from hemoglobin. For this purpose, use various options for sample preparation of material for decontamination, release from red blood cells and hemoglobin, and also use various reagents for further destruction of the microbial cell.

Thus, an analogue of this invention is a method for detecting MBT DNA by PCR in blood obtained from tuberculosis patients with extrapulmonary and pulmonary localization (Fobes B., Hicks K. // J. Clin. Microbiol. - 1993. - v.31. - p.1688-1691; Folgueira L., Delgado R., Palenque E. et al. // Neurology. - 1994.- v.44. p.1336-1338; Gamboa F., Fernandez G., Paolilla E. et al.// J. Clin. Microbiol. - 1998.- v. 36. - p. 684-689).

The detection of MBT DNA in peripheral blood in these studies varied within a fairly wide range from 50% to 85%, depending on the method of processing the material, the form of tuberculosis and the duration of treatment.

The prototype of this invention is a set of methods for processing blood and releasing the precipitate from hemoglobin, which can be used for further detection of MBT DNA by various PCR test systems.

1. As with any clinical material, there are various treatment options for blood with subsequent isolation of MBT DNA. For these purposes, the following are used: SDS, distilled H ₂ O, NaOH / NaLC, NH ₄ Cl, in various proportions with the clinical sample, at different stages of sample processing. Each of the methods of blood processing has both its advantages and disadvantages.

In most studies, the lymphocyte fraction (which is isolated using ficol) is used to isolate the MBT DNA, the plasma is not analyzed, which can lead to a certain loss of microbial cells that may be in the blood in the extracellular medium. We have shown that in cases where DNA was extracted from the lymphocyte fraction and from whole blood in the same patient, the detection of MBT in whole blood occurs in a larger percentage of cases than individually: in the lymphomonocytic fraction and blood plasma.

2. The next drawback of the methods is the collection of a large amount of venous blood, up to 20 ml (Aguado J. // Lancet 1996. - v.347. - p. 1836-1837; Folgueira L., Delgado R., Palenque E. et al. // J. Clinical Microbiology. - 1996.- p. 512-515; Gamboa F., Manterola J., Lonca J. // Int. J. Tubercle and Lung Disease. - 1997. - v. 1. - p. 542-555; Stauffer F., Haber H., Rilger A. et al. // J. Clin. Microbiol.- 1998. - v. 36. - p. 614-617; Herrington S., McGee D. // Molecular Clinical Diagnostics. Methods. M.: Mir. 1999. p.305-306.)

In all of these studies, the smallest volumes of blood used to detect MBT DNA are 5 ml. It should be noted that the larger the volume of blood, the more difficult the process of release from hemoglobin. In our studies, it was shown that in cases where the patient is a bacillus, 500-700 μl of blood is necessary and sufficient to detect DNA.

3. As systems that lyse red blood cells, we studied three types of reagents: NH ₄ Cl, NaOH / NaLC, Н ₂ О with their addition to the sample with blood in various concentrations. The best indicators (in terms of red blood cell lysing) were obtained with the following reagents: 0.085% NH ₄ Cl, (v: v blood reagent 1:10 and 2% NaOH / 0.125% NaLC 5: 1, respectively). Destroyed red blood cells with hemoglobin in the sediment after treatment with these reagents are less present than when using other treatment options. The conditions for the ratio of sample and reagent volumes were selected for the first time, and there are no analogues in the literature. As a result, the method of lysing blood erythrocytes takes the minimum time (30-40 min) compared with the methods given in the literature. The reagent is added to the blood at the desired concentration, kept for 20 minutes at room temperature and centrifuged for 10 minutes at 5000 rpm (a higher number of turns during centrifugation complicates the washing procedure from hemoglobin).

4. As systems washing off hemoglobin, various concentrations of SDS, EDTA, triton in 10 mM Tris Hcl and 1 mM EDTA (pH 7.6) and phosphate-buffered saline were studied. The best results of washing the precipitate from hemoglobin were obtained using 1% Triton in 10 mM Tris HCl buffer and 1 mM EDTA (pH 7.6). As a rule, double washing of the precipitate is sufficient to obtain a clean cell sediment.

Further heating of the precipitate in the same buffer system (pH 8.0) for 10 min at 95 ° C, followed by cooling, is sufficient for cell destruction and DNA isolation. The resulting DNA solution can be used for PCR reaction.

Thus, the proposed blood processing system, which includes the stages of the destruction of red blood cells, washing the precipitate from hemoglobin and DNA extraction in combination of the above conditions, was proposed for the first time, it is fast (processing within an hour) and effective for obtaining DNA from the office.

The differences of our proposed method from the methods used by other researchers are as follows. A smaller volume of whole venous blood (0.5 ml) is used, which is quite enough to detect MBT DNA by PCR. When working with such a volume of blood, the hemoglobin washing procedure is simplified and reduced. For the erythrocyte lysis, conventional reagents are used for the initial treatment of respiratory and some non-respiratory samples. They are freed from hemoglobin with the help of a reagent, which is further used to isolate MBT DNA and which does not interfere with the further amplification reaction. As a result, in a short time, using the same set of reagents, in one test tube it is possible to conduct sample preparation of blood samples with subsequent isolation of MBT DNA.

In order to determine the detectability of MBT DNA in blood samples by PCR, processed using these detergents, a series of experiments was carried out. Initially, blood samples with anticoagulant obtained from tuberculosis patients and healthy donors were poured into 0.5 ml tubes. To them was added a culture of M. tuberculosis H37 RV, which was bred according to the McFarland turbidity standard with a final concentration of 150 × 10 ⁶ cells / ml. After further dilution, blood samples contained M. tuberculosis in the following concentrations: 150-100; 75-50 and 10-8 cells in the sample. Further, the blood was treated according to the proposed method: 100 μl of NAOH / NaLC of the used concentration was added to 500 μl of blood, kept for 20 min at room temperature, centrifuged at 5000 rpm for 10 min, and the supernatant was discarded. Then, the precipitate was washed twice with a solution of 1% Triton in 10 mM TrisHCl and 1 mM EDTA (pH 7.6), then 20 μl of the same buffer (pH 8.0) was added and heated for 10 min at 95 ° C. Further, PCR was performed using the Nested-PCR test system (Patent No. 2163022 dated 07/04/2001), although other test systems can also be used at this stage.

The sample preparation method remains highly sensitive, since 2 μl is taken from the samples for analysis, which is equivalent to single MBT cells in terms of DNA content.

Then, studies were carried out to identify MBT DNA in peripheral blood treated with these reagents on samples obtained from patients with various forms of tuberculosis and being treated in the MNSCT. Blood samples from 42 patients were examined. Twenty-nine of them were in the active phase of the disease (up to one month of treatment), in 24 (82.7%) of which the MBT DNA was isolated from the blood.

Thus, studies have shown high detectability of MBT DNA in peripheral blood by the method of NDP in patients in the active phase of the disease.

The advantage of the proposed method, compared with those available in the literature, are the following positions:

1. A high percentage of detection (over 82% in patients in the active phase of the disease).

2. The speed and simplicity of sample preparation (1-1.2 hours and two mixtures of reagents), all operations can be carried out in one test tube.

3. Specificity, only mycobacteria are detected, since in the preparation of samples reagents are used that kill tuberculous microflora.

Claims (1)

A method of treating whole blood to detect DNA of M. tuberculosis by polymerase chain reaction, including lysis of red blood cells and washing the precipitate from hemoglobin, characterized in that a mixture of 2% NaOH / 0.125% NaLC is used as a lysing system, which is added to the blood in the blood ratio: lysis system 5: 1, the mixture was incubated for 20 min at room temperature, then centrifuged for 10 min at 5000 rpm, after which the precipitate was washed twice with 1% Triton in 10 mM Tris HCl and 1 mM EDTA buffer (pH 7.6).