RU2311640C1 - Method for isolating extracellular nucleic acids out of urine - Google Patents

Abstract

FIELD: diagnostic medicine.

SUBSTANCE: the present innovation deals with developing the ways for isolating extracellular nucleic acids (ENA) out of urine which should be applied in the course of amplification assay for earlier diagnostics of infectious and oncological diseases. For the purpose of studying the intact urine or urine treated with enzymes and/or detergents should be passed through separative-sorbing system consisting of filtering material at pores size being not more than 1.5 mcm and a fiber glass sorbent. Then one should carry out a two-stage elution of ENA connected with the surface of urinary cells being upon a filtering material due to washing the latter with a buffer solution that contains 5 mM EDTA, and then with 0.125%-trypsin solution. After that the filtering material should be removed, and the sorbent should be washed against admixtures of non-nucleotide origin to eluate the total ENA pool from the latter. The innovation enables to simplify the procedure of ENA isolation, isolate ENA out of urinary supernatant and ENA connected with the surface of urinary cells that provides the chance to increase the significance and the sensitivity of further investigation.

EFFECT: higher efficiency.

3 cl, 5 ex, 1 tbl

Description

The invention relates to the field of diagnostic medicine, namely to the development of methods for the isolation of extracellular nucleic acids (WNA) from urine, which can be used in amplification analysis for the early diagnosis of infectious and oncological diseases.

Diagnosis and detection of cancer in the early stages is an important problem in medicine. For the diagnosis of cancer using methods such as radiological, endoscopic, ultrasound, morphological. However, these methods do not allow effective detection of oncotransformation and are not effective in the early stages of the disease. In addition, morphological analysis is invasive and may affect tumor metastasis.

Known methods for the diagnosis of viral and oncological diseases by detecting DNA in plasma of peripheral blood using polymerase chain reaction (PCR) [1, 2]. The disadvantages of these methods are the invasiveness of the analysis (venous blood sampling), the high complexity and duration of DNA isolation and the low reliability of diagnostic results (34-39%) due to the low specific content of extracellular DNA in a small sample volume (limitations of the DNA extraction method), which makes it difficult clinical use of these methods.

While direct analysis of the affected tissue may be difficult or unavailable (no symptoms), and blood sampling is an invasive procedure, urine is an easily accessible biological material for the production of nucleic acids that can be used in amplification analysis.

The closest to the claimed method - the prototype, is a method for the diagnosis of kidney cancer by identifying cancer specific markers in the urine by amplification methods [3], which includes the following stages: DNA extraction from urine supernatant, detection of methylated DNA forms of tumor suppressor genes (VHL, p16, p14, APC , RASSF1A and Timp-3) by methylation-specific PCR followed by analysis of the reaction products. Extracellular DNA from the urine supernatant of patients with kidney cancer was isolated by treatment with proteinase K in the presence of sodium dodecyl sulfate overnight at 37 ° C followed by phenol / chloroform protein extraction. 1/10 volume of 10 M ammonium acetate, 2 μl of glycogen and 2.5 volumes of 100% alcohol were added to the DNA solution and incubated overnight at -20 ° C, then the sample was centrifuged at 16000 g for 20 min at 4 ° C. The supernatant was removed, the DNA precipitate was dried and dissolved in water. Isolated extracellular DNA was modified with sodium bisulfite; in amplification, primers specific for methylated forms of DNA were used. The frequency of hypermethylation of tumor suppressor gene promoters was 12% for VHL, 10% for p16, 18% for p14, 18% for APC, 52% for RASSF1A and 60% for Timp-3.

The disadvantages of this method are the duration of extracellular DNA isolation, the inconvenience of working with phenol and chloroform. In addition, this diagnostic method is time-consuming (to obtain 100% diagnostic accuracy, it is necessary to analyze promoter hypermethylation of at least 6 genes) and is applicable only for the diagnosis of kidney cancer.

An object of the invention is to increase the reliability of diagnosis, simplifying the method and expanding the scope of its use.

The technical task is achieved by the proposed method, which consists in the following.

Determine the pH of the test urine and adjust the pH to the desired value (up to 6.0-8.0 for the isolation of extracellular DNA and 4.5-5.0 for the isolation of extracellular RNA). The test urine is passed through a separation-sorption system consisting of a filter material with a pore size of not more than 1.5 microns and a fiberglass sorbent. The filter material serves to separate urine into the supernatant and the cell fraction, and the sorbent is used to sorb extracellular nucleic acids (WNA) from the urine supernatant and cell eluates. The flow of urine through the system can be reduced by centrifugation for 2-5 minutes at 400 g. Next, elution of the VNCs associated with the surface of urine cells located on the filter material is carried out by washing the latter with phosphate buffered saline (PBS) containing 5 mM EDTA and then with a 0.125% trypsin solution. In the process of elution of BHCs associated with the surface of urine cells, they simultaneously bind to the sorbent. Then the filter material is removed, and the fiberglass sorbent is washed from impurities of non-nucleotide nature. Next, the total pool of VNKs (VNKs from the supernatant of urine and VNKs eluted from the surface of urine cells) associated with the sorbent is eluted and the known specific nucleic acid sequences are detected by amplification analysis followed by PCR analysis of the products and the conclusion on the presence or absence of detectable nucleotide sequences .

The test urine can be treated with a hydrolyzing and / or denaturing agent (for example, an enzyme and / or detergent) before passing through a separation-sorbing system to break down the complexes of KSS with proteins, phospholipids, lipoproteins, etc., which leads to an increase in the efficiency of KSS excretion , and this, in turn, to increase the number of specific NK in the sample and increase the reliability of the diagnosis of the claimed method.

The use of filtering material that retains urine cells makes it possible to sequentially pass urine supernatant and eluates from the surface of urine cells through a fiberglass sorbent. As filter material, filter paper, 3MM Whatman paper, nitrocellulose material and other materials can be used.

A fiberglass sorbent binds KSS from urine supernatant and KSS eluted from the surface of urine cells. As fiberglass sorbent can be used glass wool from neutral, alkaline, borosilicate glass or mineral glass wool from basalt glass fiber containing silicon dioxide as the main component.

The use of a system containing filtering material and a fiberglass sorbent significantly simplifies, reduces the cost and speeds up the procedure for the extraction of KSS from urine as compared with the prototype, and also allows to significantly increase the volume of the test sample and, thus, increase the reliability of diagnosis by increasing the content of specific nucleic acid sequences in sample for further amplification analysis.

The defining hallmarks of the proposed method, compared with the prototype, are:

1. Passing the test urine through a system containing filter material and a fiberglass sorbent, which allows you to simultaneously separate the supernatant from the cell fraction of urine and sorb the BHC on the sorbent. The absence of filter material that retains urine cells would lead to cell destruction by passing urine through a fiberglass sorbent, as a result of which the majority of the nucleic acids released would be cell nucleic acids, which would reduce the sensitivity of the diagnostic system.

2. The use of an additional diagnostic object - VNK associated with the surface of urine cells, which allows to increase the sensitivity of detection of specific DNA and RNA sequences due to their increased specific content in the early stages of pathogenesis.

3. Sequential two-stage elution of VNCs associated with the surface of urine cells, which allows to reduce the number of ballast nucleic acids from normal cells and increase the specific content of detected specific sequences. In the prototype, specific sequences of extracellular DNA isolated from urine supernatant are analyzed.

Increasing the sensitivity of the above diagnostic system allows you to identify and differentiate all tumors of the body (tumors of the mammary gland, lung, stomach and others), while the prototype method allows you to detect only kidney cancer.

The invention is illustrated by the following examples of specific performance of the method.

Example 1

The proposed diagnostic method was tested on urine samples of healthy donors (n = 10) and patients with benign (n = 15) and malignant breast tumors (n = 20) at various stages of the disease. In all patients, urine was taken during the initial diagnosis. The pH of the test urine was checked using a litmus test and adjusted (if required) to a value of 6.0-8.0 by adding 0.1 M NaOH. Then the urine was placed in a column containing a system consisting of a paper filter and alkaline glass wool (glass fiber sorbent) and centrifuged for 2 min at 400 g. Then, the BHCs bound to the surface of urine cells located on the filter were eluted by washing the filter with an FBI filter containing 5 mM EDTA and then with a 0.125% trypsin solution. The filter was removed, the sorbent was washed twice from impurities of a non-nucleotide nature with a buffer solution containing 4.5 M guanidine thiocyanate, 20 mM EDTA, 10 mM Tris-HCl pH 6.4, and then twice with a buffer solution containing 70% ethanol, 100 mM NaCl, 10 mM Tris- HCl pH 8.0. The total pool of extracellular DNA containing KSS from the cell eluate and KSS from the urine supernatant was eluted from the sorbent by adding 100 μl of water, followed by centrifugation for 1 min at 10,000 g.

To identify the melitated sequences of the promoter regions of the RARβ2 and RASSF1A genes, methylation specific PCR was performed.

After preliminary thermal denaturation, 1 unit was added to the reaction mixture. Taq DNA polymerase (manufactured by SibEnzyme) and performed 45 cycles of PCR in the following mode:

- for the methylated form of the promoter region of the RARβ2 gene - 30 seconds at 94 ° C, 20 seconds at 65 ° C, 20 seconds at 72 ° C; primers were used [4]:

5'-CCG AAT CCT ACC CCG ACG-3 '(Primer 1);

5'-TAG TAG TTC GGG TAG GGT TTA TC - 3 '(Primer 2);

- for the unmethylated form of the promoter region of the RARβ2 gene - 30 seconds at 94 ° C, 20 seconds at 59 ° C, 20 seconds at 72 ° C; primers were used [4]:

5'-TTA GTA GTT TGG GTA GGG TTT ATT - 3 '(Primer 1);

5'-CCA AAT CCT ACC CCA ACA - 3 '(Primer 2);

- for the methylated form of the promoter region of the RASSF1A gene - 30 seconds at 94 ° C, 20 seconds at 65 ° C, 20 seconds at 72 ° C; primers were used [4]:

5'-GGG TTT TGC GAG AGC GCG - 3 '(Primer 1);

5'-GCT AAC AAA CGC GAA CCG - 3 '(Primer 2);

- for the unmethylated form of the promoter region of the RASSF1A gene - 30 seconds at 94 ° C, 20 seconds at 65 ° C, 20 seconds at 72 ° C; primers were used [4]:

5'- GGT TTT GTG AGA GTG TGT TTAG - 3 '(Primer 1);

5'- CAC TAA CAA ACA CAA ACC AAC - 3 '(Primer 2);

After 45 cycles of PCR, the products were separated on a 6% polyacrylamide gel by electrophoresis and stained with ethidium bromide.

Data on the identified melitated sequences of the promoter regions of the RARR2 and RASSF1A genes in normal and pathological conditions are presented in the table.

Genes Norm Breast Tumors, Frequency benign malignant RARβ2 0 33% 65% RASSF1A 0 53% 75% RARβ2 or RASSF1A 0 67% 95%

This example illustrates that the KSS extracted by the developed method can be used for non-invasive diagnosis of benign and malignant tumors.

Example 2

The developed method was used to evaluate the effectiveness of anti-cancer therapy in patients with lung cancer (n = 19). Urine was taken from all patients before and after anti-cancer therapy. Urine pH was checked with a litmus test and adjusted (if required) to 4.5-5.0 by adding 0.1 M acetic acid. Then the urine was placed in a column containing the system: Whatman neutral glass and centrifuged for 5 min at 400 g. To isolate RNA associated with the surface of urine cells deposited on a Whatman paper, the latter was washed with 2 ml of PBS containing 5 mM EDTA, and then 2 ml of 0.125% trypsin solution. Then whatman was removed, the sorbent was washed from impurities of a non-nucleotide nature and the total pool of extracellular RNA from the sorbent was eluted as in Example 1.

The detection of tumor RNA was performed by RT-PCR. cDNA was synthesized from random hexameric primers and the commercial Reverse Transriptase Superscript II kit (Invitrogen). For RT-PCR, the commercial kit AmplyTaq Gold (Applied Biosystems) was used using primers specific for the hTERT gene [5]:

5'-TGA CAC STS ACC TSA CCC AC-3 '(Primer 1),

5'-CAC TOT CTT CCG CAA CAA GTT CAC-3 '(Primer 2);

Next, PCR products were separated on a 2% agarose gel by electrophoresis and evaluated densitometrically using the GS-690 Imaging Densitometry System (Bio-Rad) and the Multi-Analyst / PC (Bio-Rad) computer program.

It has been shown that the hTERT marker associated with lung cancer is detected with a high frequency (95%) in patients prior to anti-cancer therapy. The patients underwent surgery followed by radiation therapy. After 4 months, we monitored the effectiveness of treatment by secondary diagnosis of cancer. The hTERT marker in the extracellular RNA pool was detected in 3 patients. Thus, the monitoring revealed the absence of cancer in 16 treated patients and the ineffectiveness of surgical and radiation treatment and the progression of cancer in 3 people.

Example 3

Patient W., born in 1975 entered the inpatient department of the Center for New Medical Technologies in Novosibirsk, with a diagnosis of gastric ulcer with the presence of Helicobacter pylori. Urine from this patient was pre-treated with an enzyme. For this, the urine was centrifuged for 10 min at 400 g, lipase (from Rhizopus japanicus 1403) was added to the obtained urine supernatant and incubated for 30 min at 37 ° C. Then the urine pH was adjusted to a value of 7.0 and passed through a column containing nitrocellulose filter material with a pore size of 1.2 μm and mineral glass wool from basalt fiberglass. The elution of the KSS associated with the surface of urine cells located on the filter, as well as the elution of the total pool of extracellular DNA from the sorbent, were carried out analogously to example 1.

The identification of Helicobacter pylori bacteria was carried out using commercial Biocom PCR reagent kits. The diagnosis has been confirmed. The patient was given conservative treatment. After 2 months, we monitored the effectiveness of treatment by secondary diagnosis on Helicobacter pylori. The monitoring revealed the absence of Helicobacter pylori. This example illustrates that the proposed diagnostic method using the KSS of urine is suitable for the detection of bacterial infections.

Example 4

Patient A., born in 1963, was admitted to the Regional Oncology Dispensary with a diagnosis of squamous undifferentiated central lung cancer with a stage of T ₂ N ₀ M ₀ . The patient was diagnosed by the claimed method using commercial Biocom PCR reagent kits (code 4.3.63), aimed at detecting the Epstein-Barr virus using the patient's urine and venous blood as a diagnostic material.

Isolation of the total pool of extracellular DNA from urine was carried out analogously to example 3, with the difference that instead of lipase, the urine was pretreated with subtilisin A.

Isolation of the total pool of extracellular DNA from blood plasma was performed by the method described in [6].

Isolation of the total pool of extracellular DNA from urine was carried out analogously to example 3, with the difference that instead of lipase, the urine was pretreated with subtilisin A.

Isolation of the total pool of extracellular DNA from blood plasma was performed by the method described in [6].

It was shown that regardless of the source of DNA, the diagnostic accuracy of the method was 100%, which indicates the possibility of using urine as a non-invasive source of diagnostic material. In addition, this example illustrates that urine extracted by the proposed method is suitable for the diagnosis of viral infections.

Example 5

Patient K., born in 1960, was admitted to the Center for New Medical Technologies in Novosibirsk, with a diagnosis of cystitis. Since women with various diseases of the genitourinary system often have mycoplasmosis, the patient was diagnosed with mycoplasma.

The test urine was centrifuged for 10 min at 400 g, sodium lauryl sulfate was added to the resulting supernatant to a final concentration of 1.0%, stirred vigorously for 2 min, and then 4 M potassium acetate was added and centrifuged for 15 min at 15000 g. Next, the supernatant pH was adjusted to 8.0 and a total pool of extracellular DNA was isolated using a system containing a paper filter and a fiberglass sorbent, as described in Example 1.

Extracellular DNA isolated from urine was used for a PNR analysis focused on the detection of mycoplasma hominis, based on the use of Biocom commercial PCR reagent kits (code 4.3.33.5). As a result of the analysis, it was shown that in the urine of patient K., Mycoplasma hominis is detected.

This example illustrates that the KNK extracted from the urine by the proposed method is suitable for the diagnosis of mycoplasma infections.

Using the proposed method will allow, in comparison with the prototype:

- to simplify the method and reduce the time of allocation of VNK from the analyzed sample to 8-10 minutes;

- increase the reliability of the subsequent diagnosis of infectious and oncological diseases due to the additional use of a more specific diagnostic indicator (VNK associated with the surface of urine cells);

exclude the receipt of false positive or false negative analysis results by increasing the volume of the sample and the number of specific nucleic acids in the sample;

- to expand the scope of application of KSS from urine due to the possibility of early diagnosis of viral, bacterial and oncological diseases, as well as the detection of mycoplasma in the body;

- increase the sensitivity of detection of specific (marker) DNA and RNA sequences associated with the surface of urine cells, which is especially important in the early stages of the development of pathologies, when the bulk of extracellular nucleic acids is associated with the surface of the cells.

INFORMATION SOURCES

1. Tailor P., Saikia T., Advani S., Mukhopadhyaya R. Activation of HHV-6 in lymphoproliferative disorders // Annanls of the New York Academy of Sciences, 2004. Vol.1022. P.282-285.

2. Sorenson G., Pribish D., Valone F., Memoli V., Bzik D., Yao S. Soluble normal and mutated DNA sequences from single-copy genes in human blood // Cancer Epidemiology, Biomarkers & Prevention, 1994. Vol. 3. P.67-71.

3. Cairns P. Detection of promoter hypermethylation of tumor suppressor genes in urine from kidney cancer patients // Annanls of the New York Academy of Sciences, 2004. Vol. 1022. P.40-43.

4. Kowara R., Golebiowski F., Chrzan P., Skokowski J., Karmolinski A., Awelczyk T. Abnormal FHIT gene transcript and c-myc and c-erbB2 amplification in breast cancer // Acta Biochimica Polonica, 2002, Vol .49. P.341-350.

5. Lonn U., Lonn S., Nilsson B., Stenkvist B. Prognostic value of erb-B2 and myc amplification in breast cancer imprints. Cancer, 1995, Vol. 75. P.2681-2687.

6. Engel E., Schmidt B., Carstensen T., Weickmann S., Jandrig B., Witt C., Fleischhacker M. Detection of tumor-specific mRNA in cell-free lavage supernatant in patients with lung cancer // Annanls of the New York Academy of Sciences, 2004. Vol. 1022. P.140-146.

7. Laktionov P., Tamkovich S., Rykova E., Vlasov V. A method for early diagnosis and monitoring of cancer. RF patent №2251696, cl. G01N 33/53, op. January 27, 2005, Bull. No. 13.

Claims (4)

1. The method of extraction of extracellular nucleic acids (WNA) from urine to identify specific nucleic acid sequences by amplification methods, characterized in that the test urine is passed through a system containing filter material with a pore size of not more than 1.5 μm and a glass fiber sorbent, a two-stage elution is performed KSS associated with the surface of urine cells located on the filter material by washing the latter with a phosphate buffer solution containing 5 mM EDTA and then with a 0.125% thrips solution on, whereupon the filter material is removed and the sorbent washed free of non-nucleotide nature of impurities and eluted with the last pool of total BHK. 2. The method according to claim 1, characterized in that the urine is pre-treated with a hydrolyzing and / or denaturing agent, in particular an enzyme and / or detergent. 3. The method according to claim 1, characterized in that mainly filter paper, Whatman 3MM, nitrocellulose are used as filter material. 4. The method according to claim 1, characterized in that as a fiberglass sorbent use glass wool from neutral, alkaline or borosilicate glass, mineral glass wool from basalt glass fiber containing silicon dioxide as the main component.