WO2015026268A1

WO2015026268A1 - Reagent allowing for microorganism inactivation, extraction and storage of bacterial dna in form suitable for highly effective molecular diagnostics

Info

Publication number: WO2015026268A1
Application number: PCT/RU2014/000616
Authority: WO
Inventors: Dmitry Alexandrovich VARLAMOV; Dmitry Garrievich SOCHIVKO; Yulia Sergeevna ALYAPKINA; Alexei Vladimirovich KUZUBOV; Larisa Nikolaevna CHERNOUSOVA; Tatyana Vladimirovna SMIRNOVA; Elena Evgenievna LARIONOVA; Sofia Nikolaevna ANDREEVSKAYA; Vladimir Georgievich Nesterenko; Viktor Veniaminovich MILITITSKY; Anatoly Petrovich SUSLOV; Natalia Yurievna MAKAROVA
Original assignee: Limited Liability Company "Nearmedic Plus"
Priority date: 2013-08-20
Filing date: 2014-08-19
Publication date: 2015-02-26
Also published as: EA201690347A1; ZA201601149B; RU2013138738A; EA032990B1

Abstract

The invention relates to biochemistry, chemistry and medicine field. A reagent for the treatment and storage of a biological sample intended for in vitro diagnostics of infectious disease via PCR method, said reagent being an aqueous solution containing an alcohol, an alkali and a tertiary amine in concentrations providing for amplification efficiency increase for at least 2 cycles, is described. The said reagent provides for neutralization of products contained in biological sample and inhibiting PCR reaction, microorganism inactivation, increase of biological sample shelf life and DNA isolation efficiency.

Description

REAGENT ALLOWING FOR MICROORGANISM INACTIVATION, EXTRACTION AND

STORAGE OF BACTERIAL DNA IN FORM SUITABLE FOR HIGHLY EFFECTIVE

MOLECULAR DIAGNOSTICS

FILED OF THE INVENTION

The invention relates to biochemistry, chemistry and medicine field and concerns development of reagent for the treatment and storage of biological samples intended for in vitro diagnostics of infectious diseases via polymerase chain reaction (PCR) method.

BACKGROUND OF THE INVENTION

At present the incidence rate of infectious diseases remains very high and their prevalence is global. Still above 1 billion of gastrointestinal and respiratory disease cases are registered every year in the world. For example, flu incidence rate in some years is above 10 to 15% population in European and American countries only. Another 75 million people suffer from other acute respiratory diseases (ARD) . During pandemics flu assumes a pattern of a natural disaster causing an enormous economic damage to countries.

Every year several tens of millions of disease cases caused by streptococci (tonsillitis, scarlet fever, erysipelas) are registered in the world. Cholera is widespread, which over the last few years is registered in more than 30 countries in the world. In the world there are more than 400 million of those suffering from trachoma and 11 million suffering from leper.

Virus hepatitis incidence rate continues to grow.

Under conditions when the most large-scale and serious

infections have shrunk the diseases which are caused by

potentially pathogenic microorganism and even usual, "normal" inhabitants of human organism have become more noticeable in the infectious pathology structure.

Over the last few years diseases earlier not regarded as such have moved into the infectious category. A group of very

peculiar "slow" infections almost unknown before is referred to. Thus, Russian investigators M. S. Margulis, V. D. Solovyev, A. K. Shubladze, E. N. Bychkova have proved a virus nature of serious central nervous diseases, human acute encephalomyelitis and disseminated sclerosis.

In accordance with the WHO information about 2 billion people, one third of the general Earth population are infected with M. tuberculosis . Currently 9 million people are tuberculized in the world each year; 3 million die from complications thereof.

Problems of combating tuberculosis, first of all, consist in early diagnostics of this disease, a well-timed treatment and carrying out a complex of preventive measures in the "nidus of tuberculosis infection" .

Current methods of laboratory diagnostics of infectious diseases do not meet the requirement of clinical practice. A fast bacterioscopy method has a low sensitivity because in order to detect tuberculosis mycobacteria (MTB) at least 100000 pathogen cells must be contained in 1 ml of pathologic material. Luminescence microscopy increases bacterioscopy sensitivity by 10 to 30% and allows for detecting 3000 to 10000 pathogen cells. Sensitivity of bacteriologic culture technique in dense

nutritious media is currently a golden standard and is about 50 to 100 MTB. However, this investigation is at least 2 months in duration.

In this connection development and practical implementation of effective methods for identification and etiologic confirmation of this disease and also other diseases acquire a high priority in modern clinical practice.

One of the most promising methods for detecting infectious disease pathogens is a method for detecting pathogen DNA via polymerase chain reaction (PCR) .

PCR diagnostics allow for identifying infectious diseases, in particular, tuberculosis several times faster than

bacteriological method. On the other hand it is possible to carry out epidemiological control of persons discharging

bacteria via PCR and thus solving issues of extending

antibacterial treatment for each specific case despite the apparent clinical X-ray stabilization of a specific process.

Detection of microorganism DNA is used in contents of a complex diagnostics for infectious diseases, allows for fast determining a presence of specific pathogen and developing effective clinical and epidemiological measures with respect to patient. PCR method provides for high sensitivity and specificity (most manufacturers of such tests guarantee sensitivity above 95%, specificity above 98%) .

In this connection the method can be used as a screening one at diagnostics of infectious disease, determining treatment policy, quality control and assessment of epidemiological importance of a patient.

Time consumed for detecting infectious agent DNA is 4 to 5 hours .

In case of massive screening of patients for infectious diseases including determination of drug resistance taking into the account implementation of automated (robotized) sample

preparation platforms the sample amount can rise up to 300 and more (an normal load of 36 samples for common bacteriological laboratory) per working day which demonstrates a possibility of concentrating large amount of biologically hazardous material in one place.

Moreover, an essential obstacle to widespread screening for infectious diseases is the fact that DNA extraction from

biological samples should be carried out during 48 hours after material sampling. This circumstance implies limitations for carrying out screening and makes material delivery and

transportation (during said time period) very costly and

difficult, especially at implementing investigations carried out in remote hardly accessible regions. In case of sampling biological samples, their delivery and subsequent storage above the aforesaid term the samples should be frozen. Freezing and thawing of samples is not allowed due to possibility of DNA destruction therein which substantially limits screening on infectious diseases even in presence of a highly specialized PCR laboratory network (available sources do not describe a method of delivery and transportation apart from freezing) .

At processing and investigation of biological material in PCR laboratories and increased risk of employee and environmental subjects infection occurs. Despite the present anti-epidemic protection measures in accordance with the current regulatory documents complex protective measures for laboratory personnel have been developed. Disease incidence of laboratory personnel carrying out tuberculosis diagnostics remains very high and it is 6 to 10 times more than the one for general population. Drug resistance of mycobacteria among anti-tuberculosis services employees is substantially higher than the drug resistance level among general population and reaches 50%. Drug resistance among other infectious diseases steadily increases according to state statistics data.

Expectoration samples received by laboratories for

investigations are characterized by high contents of infectious agent (from 5000 to 1000000 microorganisms per 1 ml of

material) . Manipulations carried out in laboratories even at the presence of protective means are doubtless risk factor for infection of medical personnel because at processing this material aerosol is formed which can contain up to 1000000 microorganisms .

Also dangerous are transportation and storage of biological material for which special safety conditions during a certain time period are required.

At present time PCR diagnostics of various pathogens in blood obtained a widespread acceptance. However even at the most serious diseases a pathogen is quite rarely determined in blood (apart from blood-transmitted infections) which demonstrates a necessity of investigating other biological materials sampled from patients in order to etiologically confirm the diagnosis . At the same time biological material samples from patients such as expectoration, biopsy materials, spinal cord liquid etc.

contains quite a lot of biological impurities such as blood, purulence, various protein substances which are, in turn, inhibitors of PCR reaction and do not allow for effectively isolating pathogen DNA.

Development of methods for preliminary preparation of biological samples allowing for subsequently carrying out effective DNA extraction based on purifying microbial cells from PCR reaction inhibitors at the step of biological sample preparation, for simultaneously inactivating microorganisms, for fluidifying the sample and carrying out this preparation by single addition of reagent to biological sample (without additional mixing steps for various ingredients) and for subsequent effective extraction of DNA, for storing and transporting the said sample during a prolonged time period in order to carry out current and further investigations (in order to determine microorganism drug

resistance) is an important problem allowing for simultaneously carrying out high-quality and fast diagnostics of infectious diseases, effective treatment of a patient and for carrying out anti-epidemiological measures directed for laboratory personnel protection.

The most difficult with respect to inactivation are tuberculosis mycobacteria. Due to specific chemical structure tuberculosis mycobacteria have a significant resistance to physical and chemical agents. In wet expectorations bacteria resist heating at 75°C for 30 min and are killed at boiling after 5 min. In isolated expectoration MTB are killed at 100°C after 45 min. In expectorations isolated and stored in darkness at room

temperature the rod viability is retained for at least 4 months, under scattered light they are killed after 1 to 1.5 month. In dry air chamber at moistening and temperature of 80°C

tuberculosis mycobacteria survive for 2 hours. In the

environment tuberculosis mycobacterium is sufficiently stable. MTB are retained in street dust up to 10 days, up to 3 months on book pages. It can be retained in water up to 150 days. Dried mycobacteria cause tuberculosis in guinea pigs after 1 to 1.5 years; lyophilized and frozen are viable up to 30 years.

There are physical and chemical methods for inactivating

microorganisms to which boiling, use of disinfectants, alcohols (ethyl and isopropyl alcohol) , quaternary ammonium bases, guanidine derivatives, aldehydes, tertiary amines should be related. MTB resistance to physical and chemical factors is determined by specifics of its outer membrane structure.

Resistance of tuberculosis pathogens to acids, alkali and alcohols is said to be linked to a lipid fraction in MTB outer membrane .

It is known that sodium hydroxide (NaOH) has a good fluidifying effect. This reagent is widely used in material sample

preparation especially that for expectorations obtained from those suffering from tuberculosis in order to fluidify it. It is known that apart from fluidifying effect alkali in a

concentration of more than 2% facilitate cell death.

Usually, purification from PCR reaction inhibitors is carried out at DNA isolation step which implies lysis of a microbial cell, DNA migration to supernatant and mixing thereof with PCR reaction inhibitors for which auxiliary methods are required in order to separate the desired DNA from other substances and its subsequent purification and concentration. In this connection existing methods for DNA isolation do not provide for sufficient sensitivity and specificity of PCR analysis.

Available sources describe various methods for inactivating microorganisms including MTB to which boiling, use of alkali, organic solvents, alcohols, detergents and disinfectants are related . Biocide detergent compositions are known based on N,N-bis-(3- aminopropyl) lauryl amine comprising N,N-bis-(3- aminopropyl) lauryl amine (EP 333143, 20.09.89) intended for disinfecting rooms and equipment but not intended for use in molecular diagnostics.

A disinfecting antimycobacterial agent is known based on N,N- bis- (3 -aminopropyl) lauryl amine and water comprising

didecyldimethylammonium chloride, C₈ - Ci₂ fatty alcohols

ethoxylates, N- (laurylaminopropylene) glycine, dodecylguanidine acetate, sodium methylenediaminetetraacetate, lauryldimethyl ammonium chloride, Ci₂ - Ci₄ fatty alcohols sulfates, decyl

sulfonate, ethanol, isopropyl alcohol apart from N,N-bis-(3- aminopropyl) lauryl amine and water (EP 343605, 29.05.89). These agents have bactericide properties, however, their use in PCR reactions is not implied; they are used only in contents of detergents at disinfecting rooms and endoscopic equipment.

An inactivating reagent has been described comprised in

GeneXpert diagnostic system. Constituents of this inactivating agent have not been disclosed. However, in the user manual and in multiple authors' publications it is said that MTB

inactivation is limited to 100 cells; full inactivation of samples is not guaranteed.

A solution is known for inactivating mycobacteria in a

biological sample comprising sodium hydroxide and isopropanol (Journal of Clinical Microbiology, Jan., 2010, pp. 229-237). Use of this inactivating reagent, as authors themselves note, does not lead to full sample inactivation but only reduces

mycobacteria concentration in the sample to > 2 CFU/ml .

A reagent is known for inactivating or decontaminating

mycobacteria in clinical samples, containing guanidine

isothiocyanate, Tris-Cl, N-lauryl sarcosyl, EDTC,

mercaptoethanol and ammonium acetate (patent RU 2338789,

20.01.08, application No. 2006124570). Use of this solution allowed the authors for achieving purity of isolated DNA. At the same time treatment of clinical samples by this solution does not lead to adequate DNA concentration in the sample which substantially reduces PCR analysis sensitivity. The described reagent is used in modified buffer of lysis which allows for carrying out sample inactivation at the present specific step. Treatment of a clinical sample by this reagent leads to lysis of mycobacterial cell and allows for optimizing DNA purification steps. In order to fluidify the sample according to present invention auxiliary steps of moderate strength alkali and mycolytic agent treatment are used. Authors do not inform about efficiency of DNA purification using the described reagent and do not indicate a possibility of using this reagent for storage and transportation of samples and also about retaining stability of the inactivated sample over time.

Therefore, literature describes multiple investigations

concerning use of various reagents for inactivating

microorganisms in biological (clinical) samples. However, none of these consider a possibility of using such a reagent for complex preparation of biological (clinical) samples for PCR investigation including purification from PCR reaction

inhibitors at the sample preparation step per se,

fluidification, possibility of safe transportation, storage of clinical samples and retaining DNA stability in the said samples during a long period of time.

Therefore, there is a need in providing a reagent for treatment and storage of a biological sample intended for in vitro

diagnostics of infectious disease, in particular, tuberculosis, by PCR method, providing for neutralization of products

contained in biological sample and inhibiting PCR reaction, microorganism inactivation, increase of biological sample shelf life and DNA isolation efficiency.

SUMMARY OF INVENTION

The present invention relates to a reagent for the treatment and storage of a biological sample intended for in vitro diagnostics of infectious disease via PCR method, said reagent being an aqueous solution containing an alcohol, an alkali and a tertiary amine in concentrations providing for amplification efficiency increase for at least 2 cycles.

Another aspect of the present invention is the reagent for the treatment and storage of the biological sample which contains the alcohol in a concentration of 32% to 41%, the alkali in concentration of 2.3% to 4.0% and the tertiary amine in a concentration of 0.01% to 0.080%. In particular, the present invention relates to the reagent wherein the alcohol is selected from a group comprising isopropanol, isobutanol and propanol; the alkali is selected from a group consisting of NaOH and KOH; the tertiary amine is selected from a group comprising

triethylamine , isopropylamine, diisopropylamine, methylethyl-c- propylamine and N, N-bis (2-aminopropyl) dodecylamine .

Yet another aspect of the present invention is the reagent which comprises 40% isopropanol, 0.2 M (2.4%) NaOH, 0.075% N,N-bis(2- aminopropyl) dodecylamine .

The invention also relates to a method for treating a biological sample intended for in vitro diagnostics of infectious disease by PCR method, said method implying treatment by said reagent during at least 1 hour.

Another aspect of the invention relates to use of said reagent for treatment and storing of a biological sample.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have prepared a reagent for the treatment and storage of a biological sample intended for in vitro

diagnostics of infectious disease, in particular, tuberculosis, via PCR method providing for neutralization of products

contained in biological sample and inhibiting PCR reaction, microorganism inactivation, increase of biological sample shelf life and DNA isolation efficiency. The said reagent can be used for treating biological samples such as expectoration, spinal cord liquid, biopsy materials and others in a form suitable for in vitro diagnostics of infectious diseases via PCR due to purification of microbial cells from PCR reaction inhibitors at the step of preparing these samples, inactivation of microorganisms including tuberculosis complex mycobacteria (MTB) in biological (clinical) sample, fluidifying of samples and retaining DNA stability in biological samples for carrying out subsequent investigations (after adding the reagent into biological material expositions during from at least 1 hour to 4 days) . A possibility of investigating this biological sample is retained up to 3 months. Moreover, storage of reagent in glass or plastic container (polypropylene) during one year provides for maintaining and stability of all reagent

constituents, for a possibility of its use during this time and also for transportation to the biological sample collection place. In connection with the use of reagent (single addition to biological material) and exposition of at least 1 hour

biological sample (especially expectoration) preparation time for PCR diagnostics is reduced at least for 2 to 3 hours.

At treatment with reagent in a biological sample both gram- negative (meningococci, honococci, veillonella, rods,

campilobacteria, helicobacteria, spirilla, spyrochaetes, rickettsia and chlamydia) and gram-positive bacteria

(pneumococci , streptococci, staphyolococci, rods, bacilli, clostridii, corynebacteria, mycobacteria, bifidobacteria and actinomyces) can be inactivated.

Comprised in the reagent are:

1. Alcohol which is selected from a group comprising

isopropanol, isobutanol and propanol. In a preferable embodiment of the invention isopropanol is used. Use of isopropyl alcohol allows for reducing surface tension at the disinfectant - microorganism cell membrane interface, for increasing

penetration of active compounds (disinfectant) into

microorganism cell structures, which, in its turn, allows for faster achievement of microorganism inactivation and using lower disinfectant agent concentrations. It is known that at equal concentrations isopropyl alcohol solution exhibits higher bactericidal activity than ethyl alcohol solution.

Alcohol can be used in a concentration of 32 to 41%, preferably, at a 40% concentration.

2. A tertiary amine which is selected from a group comprising triethylamine, isopropylamine, diisopropylamine, methylethyl-c- propylamine and Ν,Ν-bis (3-aminopropyl) dodecylamine . In a

preferable embodiment of the invention N,N-bis(3- aminopropyl) dodecylamine is used. During investigations it has been unexpectedly found that tertiary amine in combination with alcohol provides for purification of microbial cells and frees them from adhered PCR reaction inhibitors, without destructively affecting the cell wall and facilitating retaining DNA stability and simplifying its extraction upon subsequent investigation. Tertiary amine can be used in a concentration of 0.01 to 0.080%, preferably at 0.075% concentration.

3. Alkali selected from a group comprising NaOH and KOH as a fluidifying agent. In a preferable embodiment of the invention NaOH is used.

Alkali can be used in a concentration of 2.3 to 4.0%, preferably at 2.4% concentration.

Various reagent variants have been prepared (ratios of

components comprised therein were varied) and their activity was studied. Selection of quantitative ratio of components comprised in a product is within the competence of the one skilled in the art and depends on a type of a biological sample to be

investigated on presence of one or another pathogenic or

potentially pathogenic infectious agent.

Investigations carried out with various microorganisms (both gram-positive and gram-negative) have shown that effect of alcohol and alkali lead to microorganism growth delay in a biological sample and to its fluidification. However, addition of a tertiary amine has lead to an unexpected result: a

possibility of additional decontamination of samples

(purification from PCR reaction inhibitors) , retaining of cells in the sample (without lysis symptoms) , full inactivation of mycobacteria growth on nutritious media, a possibility of storing the reagent-treated biological sample during 3 months and to further effective amplification of these samples.

Mycobacteria are the most difficult microorganisms to inactivate due to structure of their cell wall. It is obvious in this connection that addition of this reagent to other microorganisms would provide a significant bacteriostatic effect and absence of microorganism growth on nutritious media.

The present inventors have also unexpectedly found that

treatment of a biological sample with reagent provides for increase in amplification efficiency at least for 2 cycles.

Investigation of activity of various reagent composition

variants

Solutions A, B, C, D with various concentrations (Table 1) and usual (traditional) method of microorganism inactivation, boiling, were used for investigation.

Table 1. Compositions of solutions used

Solution Solution composition

A Isopropanol 40%, 0.2 M (2.4%) NaOH, 0.075% (N,N,-bis- (3-aminopropyl) dodecylamine)

B Isopropanol 40%

c n-Butanol 20%

D n-Butanol 20%, 0.2 M (4%) NaOH Samples obtained from patients diagnosed with lung tuberculosis were studied. A swab was made from each sample which was stained by luminescence dyes in order to identify acid-resistant rods. Acid-resistant bacilli were identified in all samples.

Expectoration samples were divided into 5 groups which were treated with A, B, C, D solutions and boiling.

Solution A (the reagent) was the best for fluidifying

expectoration .

Various exposure times were used with solutions A, B, C, D - 1 hour, 24 hours and 4 days. Upon expiration of exposure times 0.5 ml of each fluidified and well mixed sample were sampled into a microcentrifugal test tube and used for DNA isolation; 0.5 ml were sampled into 50-ml test tube, washed 2 times with phosphate buffer and inoculated onto Middlebrook 7H9 liquid nutritious medium into MGIT tubes which were placed into BACTEC MGIT 960 device for automatic growth detection.

Expectoration from 12 bacillary patients pretreated by standard method using NALC-NaOH reagent was used as control.

Negative result (no mycobacterial culture growth) was produced after 42 days after inoculation.

Inoculation results on BACTEC MGIT 960 are outlined in Table 2.

Table 2. Results of inoculating expectoration samples treated with solutions A, B, C, D and standard NALC-NaOH reagent.

10 Growth 10 present

11 Growth 7 present

12 Growth 8 present

Solution A 1 hour 1A No growth —

2A No growth —

3A No growth —

24 hours 1A No growth —

2A No growth —

3A No growth —

4 days 1A No growth —

2A No growth —

3A No growth —

Solution B 1 hour 4B No growth —

5B No growth —

6B No growth —

24 hours 4B No growth —

5B No growth —

6B No growth —

4 days 4B No growth —

5B No growth —

6B No growth Solution C 1 hour 7C No growth —

8C No growth —

9C No growth —

24 hours 7C No growth —

8C No growth —

9C No growth —

4 days 7C No growth —

8C No growth —

9C No growth —

Solution D 1 hour 10D No growth —

11D No growth —

12D No growth —

24 hours 10D No growth —

11D No growth —

12D No growth —

4 days 10D No growth —

11D No growth —

12D No growth —

PCR results are outlined in Tables 3 to 6

Table 3. PCR results after expectoration pretreatment solution A Dye Exposure Sample # 1A Sample # 2A Sample # 3A time Average SD Average SD Average SD value (Ct) (Ct) value (Ct) (Ct) value (Ct)

(Ct)

FAM 1 hour 22.13 0.04 22.56 0.28 20.08 0.08

24 hours 21.12 0.00 21.40 0.01 19.34 0.04

4 days 19.55 0.04 20.07 0.03 17.32 0.03

ROX 1 hour 27.43 0.01 27.78 0.15 25.32 0.08

24 hours 26.31 0.06 26.71 0.06 24.51 0.10

4 days 24.78 0.01 25.36 0.01 22.37 0.06

Table 4. PCR results after expectoration pretreatment with solution B

Dye Exposure Sample # B Sample # 5B Sample # 6B

time Average SD Average SD Average SD value (Ct) (Ct) value (Ct) (Ct) value (Ct) (Ct)

FAM 1 hour 20.49 0.11 16.30 0.06 22.89 0.20

24 hours 20.86 0.07 16.91 0.10 23.31 0.21

4 days 21.00 0.30 16.82 0.33 23.38 0.01

ROX 1 hour 26.09 0.10 21.64 0.02 28.40 0.21

24 hours 26.41 0.04 22.23 0.09 28.62 0.18

4 days 26.41 0.25 22.08 0.24 28.88 0.01 Table 5. PCR results after expectoration pretreatment with solution C

Table 6. PCR results after expectoration pretreatment

solution D

Dye Exposure Sample # 10D Sample # 1 ID Sample # 12D

time Average SD Average SD Average SD (Ct) value (Ct) value (Ct) value

(Ct) (Ct) (Ct)

FAM 1 hour 21.79 0.30 20.14 0.09 19.19 0.05

24 hours 22.17 0.01 21.15 0.25 18.62 0.27

4 days 21.69 0.01 20.23 0.28 18.75 0.18 ROX 1 hour 27.13 0.18 25.37 0.10 24.21 0.01

24 hours 26.79 0.01 26.41 0.29 23.80 0.01

4 days 26.62 0.09 25.04 0.13 23.97 0.22

As a result of conducted investigations the following was established:

1. Solution A (reagent) was the best for fluidifying

expectoration .

2. Treatment of material with solutions A, B, C, D for 1 h and 24 h exposure times did not affect DNA isolation and PCR

efficiency.

3. Exposure for 4 days with A and C solutions lead to increase in DNA isolation efficiency (2 amplification cycles on the average) compared to 1 h and 24 h exposure whereas exposure with B and D solutions for 4 days did not affect PCR efficiency.

4. Treatment of expectoration with A, B, C and D solutions lead to death of mycobacterial cells at all exposure times.

5. Pretreatment with reagents is more preferable than boiling because it is a more technological method combining

fluidification, decontamination and expectoration inactivation at the same time.

At the second step a work for selecting optimal solution

component concentrations has been carried out (Table 7) , reagent volumes and sample preparation protocols for molecular-genetic investigation of expectorations from patients diagnosed with lung tuberculosis.

Table 7. Selection of solution component optimal concentrations

Sample preparation scheme for expectation samples was developed using A2 and A3 solutions; further it was compared to standard sample preparation scheme using NALC-NaOH. DNA isolation was carried out by manual and automatic method. Result was assessed according to the following criteria: expectoration

fluidification, residue formation after centrifugation, DNA yield after isolation, DNA degradation upon reagent treatment. The following sample preparation methods were studied: - Standard NALC-NaOH treatment;

- Addition of solution A in an amount of 20 to 50 ml to 3 to 5 ml volume sample;

As a result of conducted investigations of expectoration samples the following has been established:

1. A3 solution is the best for fluidifying samples. In this case an unexpected effect was noted: as the alkali concentration decreased and tertiary amine was added a more homogenous

fluidification was noted allowing for effectively carrying out the subsequent investigation steps. Moreover, alkali content in solution in an amount of 12% determines a significant corrosive effect which creates danger when working with reagent and limits its transportation. Reduction of alkali concentration to 2.4% substantially decreases this undesired property.

An experiment was carried out with native expectoration samples on pretreatment influence using A2, A5 and A5 solutions on DNA isolation via manual and automatic method. Sample preparation scheme consisted in adding A solution to samples in an amount of 20 to 50 ml. Result assessment criteria were analogous to the previous experiments. The sample preparation scheme used has demonstrated good results; solutions A2 and A5 have been

selected for further experiments.

Influence of various expectoration pretreatment methods using A2 and A5 solutions on DNA isolation and degradation compared to standard treatment procedure using NALC-NaOH were carried out on expectoration samples. The following sample preparation methods were studied:

- Standard NALC-NaOH treatment;

- Addition of solution A2 to expectoration sample in an amount up to 50 ml and mixing for 1 h;

- Addition of solution A2 to expectoration sample in an amount up to 50 ml and mixing for 1 h and incubation for 96 hours;

- Addition of solution A5 to expectoration sample in an amount up to 50 ml and mixing during 1 h;

- Addition of solution A5 to expectoration sample in an amount up to 50 ml and mixing for 1 h and incubation for 96 hours;

As a result of conducted investigations the following

conclusions have been made:

1. Pretreatment with A5 increases DNA yield and, therefore, system sensitivity compared to standard NALC-NaOH pretreatment at automatic isolation method 16.3 fold average; 11.4 fold average at manual method.

2. Incubation of samples treated with A5 for 96 hours not only does not lead to DNA degradation and decrease in yield at isolation compared to NALC-NaOH pretreatment but also

significantly increases DNA yield even compared to samples which were mixed in a presence of reagent for 1 hour without

subsequent incubation.

In order to finally select reagent composition a comparison of expectoration sample treatment with various N. tuberculosis complex DNA content treated by A2 and A5 solutions was carried out. Results are outlined in Table 8.

Table 8. Assessment of A2 and A5 reagents effect upon DNA isolation from M. tuberculosis cells at using manual and

automatic sample preparation methods.

As it is seen from the table, treatment with A5 reagent lead to a more effective DNA isolation both at manual and automatic sample preparation methods. Extremely important is an effective DNA extraction at small amount of cells in a material which is significantly higher at material treatment with A5 reagent which has been selected as the most effective.

In the following experiment a possibility of using A5 reagent has been demonstrated both on native material and on material passed through standard NALC-NaOH sample preparation; DNA isolation efficiency using the reagent being 5-10-fold higher compared to standard sample preparation.

Reagent tests for treating diagnostic material were carried out using expectoration samples from tuberculosis patients with massive bacterial excretion. Samples were split into 3 parts. First part of the samples (MTB growth control) was subjected to standard NALC-NaOH pretreatment, then cultured in liquid medium in the BACTEC MGIT 960 system. Another part of the samples was incubated for 1 h with 20 to 50 ml of reagent then washed twice with phosphate buffer and also cultured in the BACTEC MGIT 960 system. Third part of the samples was treated by standard NALC- NaOH method, 0.5 ml of reagent was added to the obtained

residues .

In no one of the investigated samples at culturing in the in the BACTEC MGIT 960 system for 42 days after incubation with A5 reagent MTB growth was identified. In control tubes without treatment with A5 reagent MTB growth was obtained. In order to understand the essence of DNA extraction improvement at PCR analysis investigation of samples after treatment with selected reagent for 1 hour was carried out. 100 expectoration samples were investigated. At examination through reverse microscope after treatment with reagent cells were "clear" from impurities (fragments "surrounding" the cell, fibers, proteins) . At that cell laceration did not occur at treatment with reagent, i.e. intracellular DNA remained within the cell and the cell itself remained free from PCR inhibiting substances which subsequently increased DNA extraction efficiency. At treating samples with reagent containing only alkali and alcohol after 1 hour exposure no effect like the one at treatment with A5 solution was

observed. At examination through reverse microscope a large number of substances "adhered" to cells was noted, especially fibers and erythrocytes.

Claims

1. A reagent for the treatment and storage of a biological sample intended for in vitro diagnostics of infectious disease via PCR method, said reagent being an aqueous solution

containing an alcohol, an alkali and a tertiary amine in

concentrations providing for amplification efficiency increase for at least 2 cycles.

2. The reagent of claim 1 which comprises the alcohol in a concentration of 32% to 41%, the alkali in concentration of 2.3% to 4.0% and the tertiary amine in a concentration of 0.01% to

0.080%.

3. The reagent of claim 1 or claim 2 wherein the alcohol is selected from a group comprising isopropanol, isobutanol and propanol, the alkali is selected from a group consisting of NaOH and KOH, the tertiary amine is selected from a group comprising triethylamine, isopropylamine, diisopropylamine, methylethyl-c- propylamine and Ν,Ν-bis (3-aminopropyl) dodecylamine .

4. The reagent of claim 1 or claim 2, which comprises 40% isopropanol, 0.2 M (2.4%) NaOH, 0.075% N,N-bis(3- aminopropyl) dodecylamine .

5. A method for treating a biological sample intended for in vitro diagnostics of infectious disease via PCR method, said method implying treatment with the reagent according to any one of claims 1 to 4 for at least 1 hour.

6. Use of the reagent according to any one of claims 1 to 4 for treating and storing of a biological sample.