KR100546713B1 - Extraction method of dna from microorganism for pcr - Google Patents

Abstract

 The present invention relates to a method for extracting microbial DNA for PCR testing. In particular, the present invention comprises the addition of a PCR buffer containing a non-ionic surfactant or a proteinase K-resin solution to the microbial cells or microbial culture, and heat-treating it to obtain a supernatant containing DNA As a microbial DNA extraction method for PCR, a DNA extraction method comprising adding mineral oil at 0.2 to 10 times by volume to the mixture after adding the PCR buffer or proteinase K-resin solution or at the same time By doing so, the sensitivity of the PCR test can be significantly improved, and microbial infection can be accurately diagnosed.

Mycobacterium tuberculosis, diagnostics, PCR, DNA extraction, mineral oil

Description

Microorganism DNA Extraction Method for PCR Testing {EXTRACTION METHOD OF DNA FROM MICROORGANISM FOR PCR}

Figure 1 is an electrophoresis picture showing the results of the PCR after performing the isolated DNA of Example 1 as a template.

Figure 2 is an electrophoresis picture showing the results of the PCR after the PCR of the isolated DNA of Comparative Example 1 as a template.

Figure 3 is an electrophoresis picture showing the results of PCR after performing the template of the separated DNA of Example 2.

Figure 4 is an electrophoresis picture showing the results of the PCR after performing the isolated DNA of Comparative Example 2 as a template.

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a method for extracting microbial DNA for PCR, and more particularly, to a method for extracting microbial DNA that can significantly diagnose the microbial infection by significantly improving the sensitivity of a PCR test.

[Private Technology]

Tuberculosis is an infection caused by the inhalation of small droplets of the tuberculosis bacteria, Mycobacterium tuberculosis , into the lungs. It is a chronic infectious disease that has been studied the most as a single disease in medical history. Tuberculosis morbidity and mortality have been greatly reduced due to the development of anti-tuberculosis chemotherapy and active anti-tuberculosis business, but tuberculosis has been reactivated as the number of patients with acquired immunodeficiency syndrome has recently increased. There are many cases of simultaneous or concurrent infections, and with the advent of multidrug-resistant tuberculosis bacteria, interest in tuberculosis is heightened (Bloom, BR and Murray, CJ, 1992; Nowak, R., 1995). In Korea, the prevalence of active tuberculosis revealed by radiographic examination in 1995 was 1.0%, which is estimated to reach 430,000 tuberculosis patients. In recent years, the rate of tuberculosis patients has slowed, but at the same time, 30,000 new patients are registered annually.

The main diagnostic methods for tuberculosis bacteria are chest radiography, sputum acid staining and tuberculosis culture. Anti-acid staining method is simple and rapid, while the specificity is low and the sensitivity is very low compared to the tuberculosis culture test method, there is a problem that the detection limit is only 1 x 10 ⁴ number of tuberculosis bacteria per 1 ml of sample (De Wit, D. et al. , 1990; Forbes, BA and Hicks, KE, 1993). The tuberculosis culture test is used for the diagnosis of tuberculosis, it is possible to detect 10 to 100 or more live bacteria positively per 1 ml of the sample is relatively high sensitivity, and has the advantage of being able to perform the sensitivity test for antibiotics. However, it takes about 6-8 weeks to incubate, and the diagnosis time is long, and during this period, the patient has a high risk of infecting tuberculosis with others (Tsukamura, M., 1981). In order to solve the above problems a study to develop a more rapid and accurate diagnosis is in progress, and (Pao, CC et al., 1990), 14 the growth of bacteria by the addition of C- labeled palmitate in the culture medium CO ₂ BACTEC system that can be detected in a short time by increasing (Ellner, PD et al. , 1988), Enzyme immunization with monoclonal antibodies (Schoningh, R. et al. , 1990) and hybrid binding assays with DNA markers (Cousins, DV et al. , 1992) have been developed. However, the method requires an expensive tester and reagents, and has a disadvantage in that a culture period for securing a sufficient amount of bacteria required for the test is required.

In addition, because clinical samples contain very small amounts of bacteria, molecular diagnostics with high sensitivity and specificity, such as strand displacement amplification (SDA), polymerase chain reaction (PCR), and reporter phage ) Can be analyzed. Among them, the PCR method has been evaluated as a very useful means for diagnosing microbial infectious diseases because of its excellent sensitivity, rapidity and specificity. Eisenach et al. (1990) reported that up to one tuberculosis bacterium could be detected by amplifying a portion of IS 6110, an insertion-like sequence that is present in 10-16 copies within the Mycobacterium tuberculosis chromosome. have.

The detection of Mycobacterium tuberculosis in clinical specimens by PCR can be divided into isolation of Mycobacterium tuberculosis in the sample, isolation of Mycobacterium tuberculosis DNA from the isolates, PCR and product analysis. The detection result by the double PCR method depends on the pretreatment method of the specimen including the isolation of Mycobacterium tuberculosis and the isolation of DNA. Therefore, the pretreatment of clinical specimens is very important for the accuracy of detection results and the reduction of diagnosis time and cost.

In order to solve the problems of the prior art, an object of the present invention is to provide a microbial DNA extraction method for PCR testing that can improve the sensitivity and specificity of microbial detection when detecting microorganisms using PCR. .

In addition, an object of the present invention is to provide a microbial DNA extraction method for PCR test that can be diagnosed whether or not microbial infection by simply and quickly examine a large amount of samples.

It is another object of the present invention to provide a composition for extracting microbial DNA for PCR testing.

In order to achieve the above object, the present invention provides a PCR test comprising adding a PCR buffer containing a nonionic surfactant to a microbial cell or a microbial culture microbial suspension and heat-treating it to obtain a supernatant containing DNA. As a microbial DNA extraction method, it provides a DNA extraction method comprising mixing the mineral oil 0.2 to 10 times by volume with respect to the mixture after the PCR buffer addition or at the same time.

In another aspect, the present invention is a PCR test comprising the addition of the Killex 100 (chelex-100) and proteinase K to the microbial cells or bacterial suspension of the microbial culture, and heat treatment it to obtain a supernatant containing DNA As a microbial DNA extraction method, it provides a DNA extraction method comprising mixing the mineral oil in 0.2 to 10 times by volume with respect to the mixed solution after the addition of the Killex 100 and proteinase K.

The present invention also provides a microbial DNA extraction composition for PCR testing comprising a nonionic surfactant, PCR buffer and mineral oil.

In another aspect, the present invention provides a microbial DNA extraction composition for PCR test containing Killex 100, proteinase K and mineral oil.

In another aspect, the present invention (a) extracting the microbial DNA by the microbial DNA extraction method for the PCR test, (b) performing the PCR using the DNA as a template, a primer for amplifying the microbial DNA and (c) the After PCR, the presence or absence of amplification of the microbial DNA provides a microbial detection method comprising determining that the microorganism is present when amplified.

Hereinafter, the present invention will be described in detail.

The present inventors have been investigating ways to improve the detection limit in diagnosing the infection of microorganisms in the sample by PCR method, and confirmed that the PCR detection efficiency is significantly increased when mineral oil is added in the sample pretreatment step before PCR. Based on this, the present invention was completed.

The present invention relates to a microbial DNA extraction method and extraction composition for PCR.

The microbial DNA extraction method for PCR testing of the present invention can be carried out on all kinds of microorganisms, preferably bacteria, fungi, yeasts or viruses, and more preferably humans, animals or plants to infect diseases Causing pathogenic bacteria, fungi or viruses, most preferably M. tuberculosis , bovine tuberculosis ( M. bovis ), mold tuberculosis ( M. avium ), M. Khan sasiyi (M. kansasii), M. Marie num (M. marinum), M. Le fry (M. leprae), when Pula to M. disk Stadium (M. scrofulaceum), M. potuyi Tomb (M. fortuitum ), M. ulseo lance (M. ulcerans), M. cello age (chelonai M.), M. intra three Lula Li (M. intracellulare), M. ply (M. phlei), M. Smeg clematis (M. smegmatis ) , M. gordonae , M. xenopi , M. gastri , M. terrae , M. smiae , M. Malmo Enschede (M. malmoense), M. Plastic chopping CL (M. flavescens), M. seujul Guy (M. szulgai) Mycobacteria genus strains, such as acid-fast bacteria and the like, and anti- acid fungi such as strains of Nocardia genus or Rhodococcus genus.

Microbial DNA extraction method for PCR test of the present invention, conventional boiling method (boiling method, Seo Soon-pal et al. , 1995; Buck, GE et al. , 1992; Imjin, et al. , 1997) or proteinase K-resin treatment method (Seo, Dae-young, etc.) , 1997; Walsh, PS et al. , 1991; Singer-Sam, J. at al. , 1989), and the process of mineral oil addition.

Specific microbial DNA extraction method for PCR testing, (a) simultaneously or sequentially adding a PCR buffer containing a nonionic surfactant and mineral oil to the microbial cells or bacterial suspension of the microbial culture and (b) heat treatment the mixture And then obtaining a supernatant containing DNA.

The nonionic surfactant may be a conventional nonionic surfactant, and is preferably a nonionic surfactant used in DNA extraction for PCR testing. Examples include Triton X-100, Nonidet 40 and Tween 20, which may be used alone or in mixture of two or more thereof. The use amount of the nonionic surfactant may be 0.30 to 1.50 (v / v)% in the PCR buffer, but is not limited thereto.

The PCR buffer can be appropriately adjusted according to the characteristics of the DNA to be amplified, such as the G + C ratio or the position of the amplification target, or the PCR temperature conditions, and can be particularly used at a composition ratio described in known literature. In one example, the PCR buffer comprises Tris-HCl and one of KCl, NaCl or (NH ₄ ) ₂ SO ₄ and MgCl ₂ or MgSO ₄ and may further comprise gelatin. The composition ratio is 5.0 to 50 mM Tris-HCl pH 7.5 to 9.0, 10 to 100 mM KCl or NaCl or (NH ₄ ) ₂ SO ₄ , 1.0 to 7.5 mM MgCl ₂ or MgSO ₄ and 0.00001 to 0.01 (w / v )% Gelatin, but is not limited thereto. PCR buffer may be added at 20 to 1000 μl per 100 μl sample.

The mineral oil (CAS # 8020-83-5; Sigma cat # M5904) can be used by purchasing a commercially available product, and in general, a middle layer on the upper sample for the purpose of preventing surface drying of the PCR sample during PCR. It may be a mineral oil. Mineral oil may be used in a mixed solution of the sample sample and the PCR buffer 0.2 to 10 times by volume, but is not limited thereto.

The heat treatment may be performed at 80 to 100 ° C. with a hot water bath, and the hot water time may be, for example, 5 to 60 minutes, but is not limited thereto. After the heat treatment, the heat treatment may be centrifuged, for example, by centrifugation at 6000 to 15000 xg, and the supernatant may be obtained to prepare a DNA sample for PCR.

The conditions employed in the microbial DNA extraction method for PCR described above, in particular, the PCR buffer composition, the content and type of the non-ionic surfactant can be easily modified by those skilled in the art to which the present invention pertains. All of them are included in the scope of the present invention.

In addition, the microbial DNA extraction method for PCR testing of the present invention, (a) adding the Chelex 100 (chelex-100) and proteinase K simultaneously or sequentially with mineral oil to the microbial cells or microbial culture suspension And (b) heat treating the mixture to obtain a supernatant containing DNA.

Since the Killex 100 and the proteinase K are conventionally used for extracting microbial DNA, a commercially available product can be purchased and used, and the amount of use can also be used according to a known technique. For example, Chelex 100 (chelex-100: Bio-Rad Laboratories) is used in the microbial cells or culture suspension of the microbial suspension of 0.1 to 5 times by volume, and the Killex 100% stock solution or dilution of 2 to 20 (w / v)% use. Proteinase K is used at 0.05 to 1.0 (w / v)% in microbial cells or bacterial suspension of culture.

Addition of the mineral oil and step (b) are the same as described above.

The supernatant containing DNA prepared by the method for extracting microbial DNA for PCR according to the present invention is used as a sample of a PCR reaction, and PCR is performed. PCR can be easily carried out according to a known technique.

In one embodiment, the present invention provides a microbial detection method using PCR. The method for detecting Mycobacterium tuberculosis may include (a) extracting microbial DNA by the microbial DNA extraction method for PCR according to the present invention, (b) performing PCR using the microbial DNA amplification primer as a template, and ( c) after the PCR, and confirming the presence or absence of amplification of the microbial DNA, and determines that the microorganism is present when amplified. The microorganism may be any conventional detectable species, for example tuberculosis bacteria. The primer may be a specific primer known according to each microorganism, for example, in the case of Mycobacterium tuberculosis, the primer is a primer capable of specifically detecting an IS 6110 insertion sequence, and includes oligonucleotides of SEQ ID NOs: 1 and 2, for example. It may be a primer. Amplification of the microbial DNA can be easily confirmed by electrophoresis, but is not limited thereto.

In one embodiment of the present invention, DNA was obtained by DNA extraction with the addition of mineral oil according to the present invention from Mycobacterium tuberculosis, and then PCR was performed to determine a detection limit detected by PCR. In addition, unlike the DNA extraction method of the present invention as a comparative group, Mycobacterium tuberculosis DNA was obtained by a known boiling method and a proteinase K-resin treatment method without adding mineral oil, and then the same PCR was performed. As a result, it was confirmed that the detection limit of the method of the present invention is improved by 10 times or more compared to the comparison group. Therefore, the DNA extraction method of the present invention significantly increases the sensitivity of microbial detection by PCR, thereby increasing diagnostic accuracy.

The present invention also provides a microbial DNA detection composition for PCR testing. The microbial DNA detection composition for PCR test includes a nonionic surfactant, PCR buffer and mineral oil. Preferably the composition comprises a nonionic surfactant, PCR buffer and mineral oil in a volume ratio of 1: 150 to 250: 400 to 800. The nonionic surfactant may be a conventional nonionic surfactant, and is preferably a nonionic surfactant used in DNA extraction for PCR. Examples include Triton X-100, Nonidet 40 and Tween 20, which may be used alone or in mixture of two or more thereof. The PCR buffer can be appropriately adjusted according to the characteristics of the DNA to be amplified, such as the G + C ratio or the position of the amplification target, or the PCR temperature conditions, and can be particularly used at a composition ratio described in known literature. In one example, the PCR buffer comprises Tris-HCl and one of KCl, NaCl or (NH ₄ ) ₂ SO ₄ and MgCl ₂ or MgSO ₄ and may further comprise gelatin. The composition ratio is 5.0 to 50 mM Tris-HCl pH 7.5 to 9.0, 10 to 100 mM KCl or NaCl or (NH ₄ ) ₂ SO ₄ , 1.0 to 7.5 mM MgCl ₂ or MgSO ₄ and 0.00001 to 0.01 (w / v )% Gelatin, but is not limited thereto.

Another microbial DNA detection composition for PCR testing includes Killex 100, Proteinase K and mineral oil, preferably, Killes 100, Proteinase K and mineral oil 1: 0.005 to 0.1: 5 to 100 weight ratio It can be included as. Since the Killex 100 and the Proteinase K are conventionally used for extracting microbial DNA, a commercially available product can be purchased and used, and the Killex is a stock solution or a dilution of 2 to 20 (w / v)%. Can be used as

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example 1

A standard tuberculosis strain ( M. tuberculosis H37Rv ATCC 27294), 2 weeks old, in Lowenstein-Jensen medium (TB, Korea) was purchased from the tuberculosis researcher.

Standard tuberculosis strains cultured in Lowenstein-Jensen medium for 2 weeks were placed in 5 ml of PBS (pH 7.4), and the cells were suspended and washed, and centrifuged at 2,500 xg for 5 minutes. The precipitate was resuspended in PBS (pH 7.4) to give McFarland No. Dilute to a haze of 0.5 (cell concentration 1.5 × 10 ⁹ / ml). Diluted 10 times each step, and then the standard bacterial suspension to be 10 ⁰ , 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 , 10 ^-8 times Was prepared.

200 μl of PBS (pH 7.4) was added to 100 μl of the standard Mycobacterium tuberculosis suspension, followed by centrifugation at 12,000 × g for 5 minutes to remove 200 μl of the supernatant. Into 100 μl of remaining acupuncture solution, 50 μl of PCR buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl ₂ , 0.001% gelatin) containing 0.45% Triton X-100 and 0.45% Tween 20, 150 μl of mineral oil (CAS # 8020-83-5; Sigma cat # M5904) was added thereto, followed by agitation at 100 ° C. for 30 minutes to release the DNA, followed by shaking for 30 seconds. After centrifugation at 12,000 xg for 30 seconds, 50 µl of the supernatant was taken and refrigerated and used for PCR.

Example 2

In the same manner as in Example 1, except that DNA separation from the standard Mycobacterium tuberculosis suspension was performed by the following method.

200 µl of PBS (pH 7.4) was added to 100 µl of the standard Mycobacterium tuberculosis suspension, followed by centrifugation at 12,000 xg for 5 minutes to remove 200 µl of the supernatant. 200 µl of 5% resin (Chelex-100: Bio-Rad Laboratories) and 5 µl of 20 mg / ml proteinase K were added to 100 µl of the remaining sap solution, and 150 µl of mineral oil was added, followed by 3 Incubated for hours. Thereafter, the mixture was agitated at 100 ° C. for 10 minutes to liberate DNA, and then shaken vigorously for 30 seconds, followed by immersion for 3 minutes at 12,000 × g. 50 μl of the supernatant was used for PCR.

Comparative Example 1

Standard Mycobacterium tuberculosis suspension was prepared in the same manner as in Example 1, and DNA separation from the bacterial suspension was performed by a boiling method (Seo Soon-pal et al. , 1995; George E. Buck. Et al. , 1992; Imjin et al. , 1997 ).

100 μl of the standard Mycobacterium tuberculosis suspension or 200 μl of PBS (pH 7.4) was added to the suspension of the sputum sample, followed by centrifugation at 12,000 × g for 5 minutes to obtain 100 μl of the salivary solution. 50 μl of PCR buffer (10mM Tris-HCl pH 8.3, 50mM KCl, 1.5mM MgCl ₂ , 0.001% gelatin) containing 0.45% Triton X-100 and 0.45% Tween-20, the cell lysis buffer After that, DNA was liberated by 30 minutes of hot water at 100 ° C., centrifuged at 12,000 × g for 20 seconds, and 50 μl of the supernatant was refrigerated and used for PCR.

Comparative Example 2

A standard tuberculosis suspension was prepared in the same manner as in Example 1, and DNA separation from the bacterial suspension was carried out using the protease K-resin method (S. Dae et al. , 1997; Walsh, PS et al. , 1991; Singer-Sam, J. et al. , 1989).

200 μl of PBS (pH 7.4) was added to 100 μl of standard Mycobacterium tuberculosis suspension or suspension of sputum sample, and then 200 μl of the supernatant was removed by centrifugation at 12,000 × g for 5 minutes. To 100 μl of remaining sap, 200 μl of 5% resin (Chelex-100: Bio-Rad Laboratories) and 5 μl of 20 mg / ml proteinase K were added. After incubation for 3 hours in a 55 ℃ constant temperature water bath, the DNA was liberated by agitation at 100 ℃ for 10 minutes, and centrifuged for 3 minutes at 12,000 xg. 50 μl of the supernatant was taken, refrigerated and used for PCR.

Experimental Example 1: detection of Mycobacterium tuberculosis by PCR

1. Primer

Primer sets (SEQ ID NOs: 1 and 2) for amplifying the IS 6110 insertion sequence (SEQ ID NO: 1 and 2) which are specifically present in the DNA of Mycobacterium tuberculosis were commissioned by Korea Bioneer (Korea). The size of the PCR product amplified with the primer set is 304 bp.

2. PCR

The reaction mixture of PCR was 50 mM KCl, 1.5 mM MgCl ₂ , 10 mM Tris-HCl (pH 8.3), 200 uM (each) -dNTPs (dATP, dTTP, dGTP and dCTP), 2 pmol primer set each, 1U Taq 10 μl of the polymerase (Takara r-Taq; Takara, Japan) and DNA solution were mixed and titrated to a total volume of 60 μl. The amplifier was a Perkin-Elmer 9600 (Perkin-Elmer, USA), the PCR reaction was 95 ℃, 3 minutes-> (95 ℃, 30 seconds-> 72 ℃, 2 minutes 15 seconds), 40 repetitions-> 72 C, it was 3 minutes.

3. PCR analysis and results

After PCR, 10 µl of the reaction product was taken, and 2 µl of 6x gel loading buffer (0.25% bromphenol blue, 0.25% xylene cyanol FF, 30% glycerol) was mixed, followed by 2% agarose gel (Seakem LE Agarose: CAMBREX, USA) was electrophoresed at 100V for 30 minutes. After dyeing with 0.1% EtBr (ethidium bromide) for 15 minutes, the photo was taken with UV (wavelength 302 nm) transluminometer (UVP SL-20, USA) and gel photographer RAISER RA 1 (Vilber Lourmat, Germany). The amplified band size was confirmed. 100 bp DNA ladder (Invitrogen Life Technologies, USA) was used as a DNA size marker during electrophoresis.

Figure 1 is an electrophoresis picture showing the results of the PCR after performing the PCR with the template of Example 1, was able to detect the tuberculosis DNA by PCR to a concentration of standard bacteria suspension diluted 10 ^-6 times.

Figure 2 is an electrophoresis picture showing the results of PCR after performing the PCR with the template of Comparative Example 1, Mycobacterium tuberculosis DNA was detected by PCR to a concentration of standard bacteria suspension diluted 10 ^-5 times. This shows that the detection limit is lower than that of the PCR result of Example 1, which separates DNA in the same manner as Comparative Example 1 but uses mineral oil during the separation process.

Figure 3 is an electrophoresis picture showing the results of PCR after performing the PCR with the template of Example 2, was able to detect the tuberculosis DNA DNA by PCR to a concentration of standard bacteria suspension diluted 10 ^-7 times.

Figure 4 is an electrophoresis picture showing the results of PCR after performing the PCR with the template of Comparative Example 2, Mycobacterium tuberculosis DNA was detected by PCR to a concentration of standard bacteria suspension diluted 10 ^-6 times. This shows that the detection limit is lower than the PCR result of Example 2 which separates DNA in the same manner as Comparative Example 2 but uses mineral oil during the separation process.

The results of FIGS. 1 to 4 are collectively shown in Table 1 below. In Table 1, it can be seen that the DNA separation method using mineral oil is significantly superior to DNA separation efficiency and PCR detection limit compared to the conventional method. Therefore, when PCR is performed by separating DNA by the method of the present invention, the PCR detection limit can be improved by about 10 times.

M. tuberculosis H37Rv PCR results for diluents 10 ⁰ 10 ^-1 10 ^-2 10 ^-3 10 ^-4 10 ^-5 10 ^-6 10 ^-7 10 ^-8 Comparative Example 1 + + + + + + - - - Comparative Example 2 + + + + + + + - - Example 1 + + + + + + + - - Example 2 + + + + + + + + -

Example 3

Of the 48 patients who were diagnosed with tuberculosis on X-ray at Milyang Yeungnam Hospital and treated with Mycobacterium tuberculosis culture in the clinical pathology and microbiological laboratory, 48 sputum specimens were examined.

To the sputum specimens commissioned by the tuberculosis culture test in the clinic, the same amount of 1M NaOH was added, mixed on a shaker and decontaminated at room temperature for 20 minutes. PBS (pH 7.4) was added thereto, titrated to a final volume of 50 ml, centrifuged at 2,500 xg for 20 minutes, and the supernatant was carefully discarded, and about 1 ml of the precipitate was resuspended in PBS. 100 μl each of the suspension was used for anti-acid staining and Mycobacterium tuberculosis culture, and the rest was immediately refrigerated until DNA extraction (Sung Joon et al., 1993).

The suspension was treated in the same manner as in Example 1 to separate DNA.

Example 4

The same sample as in Example 3 was carried out in the same manner, except that DNA was separated from the suspension by the method of Example 2.

Comparative Example 3

The same sputum specimen suspension was used as the sample in Example 3, and DNA was isolated by the same method as in Comparative Example 1.

Comparative Example 4

The same sputum specimen suspension was used as the sample in Example 3, and DNA was isolated by the same method as in Comparative Example 2.

Experimental Example 2: Sensitivity and Specificity Test of Mycobacterium Tuberculosis According to Test Methods

Clinical specimens were tested for culture, AFB (Acid Fast Bacilli, Ziehl-Neelsen staining), and PCR, respectively, to determine the sensitivity and specificity of the detection of Mycobacterium tuberculosis.

The clinical specimens were sputum 48 specimens of tuberculosis bacteria tested in clinical pathology and microbial laboratory among patients who were diagnosed with tuberculosis on X-ray at Milyang Youngnam Hospital.

(1) AFB test method

Sputum specimens were tested for tuberculosis infection by Ziehl-Neelsen staining. 100 [mu] l of the suspended solids decontaminated with 1 M NaOH was taken, smeared on a slide, dried and fixed with flame. Covered with the dye solution Carbolfuchsin (0.3% basic fuchsin, 95% ethyl alcohol, 5% phenol) and heated with a flame to leave steaming for 5 minutes and washed with water. After bleaching with 3% HCl alcohol bleaching solution for 2 minutes, washing with water and staining with 0.3% methylene blue control dye for 30 seconds. After washing with water and dried, the whole field of vision plated at 1,000 times was microscopically examined. If positive acid bacterium (AFB: Acid Fast Bacilli) was seen, the result was positive.

(2) culture test method

100 μl of the suspension contaminated with 1M NaOH was inoculated onto the slope of 3% Ogawa medium (EIKEN, Japan), and then the cap was loosely closed and placed horizontally in a 37 ° C. incubator overnight. The next day, the lid was closed tightly, and the medium was raised and incubated in a 37 ° C. incubator once a week for 8 weeks. If colony appeared during the observation period, it was judged as positive, and if not, negative.

In addition, to distinguish between Mycobacterium tuberculosis and Mycobacterium Other Than Tuberculosis (MOTT), a niacin test was performed. For the strains grown in Mycobacterium tuberculosis culture, on the slope of 3% Ogawa medium, 1.5 ml of sterile water (DW) or saline was added to the surface of the colonies where the antibacterial bacteria were concentrated and colonized with sterile platinum. The surface was broken up to allow the liquid to contact the medium. The liquid was developed at room temperature for 20-30 minutes while covering the surface of the bacterial colonies to allow niacin to be extracted. Then niacin strip (BBL, USA) was added to observe the color change. According to the presence or absence of color, it was negative if there was no color change and positive if yellow.

(3) PCR test method

Example 3 and 4 and the separated DNA of Comparative Examples 3 and 4 were analyzed by PCR in the same manner as in Experiment 1.

The analysis results through the above experiments are shown in Tables 2 and 3 below.

Standard Culture Assay Experiment result (+/-) AFB test method PCR assay Comparative Example 3 Comparative Example 4 Example 3 Example 4 +: 18 11/7 12/6 14/4 15/3 18/0 -: 30 0/30 1/29 1/29 1/29 1/29

Comparative Example 3 Comparative Example 4 Example 3 Example 4 responsiveness 66.7% (12/18) 77.8% (14/18) 83.3% (15/18) 100% (18/18) Specificity 96.7% (29/30) 96.7% (29/30) 96.7% (29/30) 96.7% (29/30) Positive predictive index 92.3% (12/13) 93.3% (14/15) 93.8% (15/16) 94.7% (18/19) Speech prediction index 82.9% (29/35) 87.9% (29/33) 90.6% (29/32) 100% (29/29) Sensitivity = (test protons / patients) x 100 (%) specificity = (test negatives / non-patients) x 100 (%) positive predictive index = (test positive disease / test protons) x 100 ( %) Negative Predictive Index = (Negative Patients Tested / Number of Tested Patients) x 100

In Table 2, the detection sensitivity of Mycobacterium tuberculosis from clinical specimens was better than that of the AFB assay, and the PCR assay was able to measure the infection of Mycobacterium tuberculosis in a short time compared to the culture test which took a long time.

In Table 3, it can be seen that the method of Examples 3 and 4 of the present invention showed significantly improved sensitivity and positive and negative predictive indexes compared to Comparative Examples 3 and 4.

As described above, the microbial DNA extraction method for PCR test of the present invention significantly improves the sensitivity of the PCR test, which can quickly and simply test a large amount of samples. Therefore, the present invention can accurately diagnose whether the microorganism is infected, and can effectively prevent secondary infection by pathogenic microorganisms.

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Claims (10)

A microbial DNA extraction method for PCR testing comprising adding a PCR buffer containing a nonionic surfactant to a microbial cell suspension or microbial culture microbial suspension, and heat-treating it to obtain a supernatant containing DNA. DNA extraction method comprising mixing the mineral oil in 0.2 to 10 times by volume with respect to the mixture after the PCR buffer addition or at the same time. The method of claim 1, wherein the microorganism is M. tuberculosis ( M. tuberculosis ), M. bovis ( M. bovis ), M. avium ( M. avium ), M. Kansasi ( M. kansasii ) , M. marinum , M. scrofulaceum , M. fortuitum , M. ulseo lance (M. ulcerans), M. cello age (chelonai M.), M. intra three Lula Li (M. intracellulare), M. ply (M. phlei), M. Smeg clematis (M. smegmatis ) , M. gordonae , M. xenopi , M. gastri , M. terrae , M. smiae , M. Malmo Enschede (M. malmoense), M. Plastic chopping CL (M. flavescens), M. seujul guide (M. szulgai), made of a no-carboxylic Dia (Nocardia) in strain and Rhodococcus (Rhodococcus) sp Selected from the group. The method of claim 1, The nonionic surfactant is selected from the group consisting of Triton X-100, Nonidet 40, Tween 20 and mixtures thereof; Said PCR buffer comprises (a) Tris-HCl, (b) one compound selected from the group consisting of KCl, NaCl and (NH ₄ ) ₂ SO ₄ and (c) MgCl ₂ or MgSO ₄ ; The heat treatment is carried out at 80 to 100 ° C. for 5 to 60 minutes; The supernatant containing the DNA is obtained by centrifugation for 10 seconds to 30 minutes at 6000 to 15000 xg. Microbial DNA extraction method for PCR testing comprising the addition of Killex 100 (chelex-100) and proteinase K to the microbial cells or microbial culture of the microbial culture suspension, and heat-treating it to obtain a supernatant containing DNA As DNA extraction method comprising mixing the mineral oil in 0.2 to 10 times by volume with respect to the mixed solution after the addition of the Killex 100 and proteinase K. The microorganism of claim 4, wherein the microorganism is M. tuberculosis , M. bovis , M. avium , M. kansasi (M. tuberculosis). M. kansasii ) , M. marinum , M. scrofulaceum , M. fortuitum , M. ulseo lance (M. ulcerans), M. cello age (chelonai M.), M. intra three Lula Li (M. intracellulare), M. ply (M. phlei), M. Smeg clematis (M. smegmatis ) , M. gordonae , M. xenopi , M. gastri , M. terrae , M. smiae ), M. in Malmo Enschede (M. malmoense), M. Plastic chopping CL (M. flavescens), M. seujul guide (M. szulgai), no carboxylic Dia (Nocardia) in strain and Rhodococcus (Rhodococcus) sp The method is selected from the group consisting of. The method of claim 4, wherein The reaction after adding the Chelex 100 and the proteinase K is carried out at 35 to 60 ° C. for 1 to 30 hours; The heat treatment is a water bath at 80 to 100 ° C. for 5 to 60 minutes; The supernatant containing the DNA is obtained by centrifugation for 10 seconds to 30 minutes at 6000 to 15000 xg. delete delete delete (a) extracting the microbial DNA by the microbial DNA extraction method for PCR test according to any one of claims 1 to 6; (b) performing PCR using the DNA as a template and a primer for amplifying microbial DNA; And (c) checking the presence or absence of amplification of the microbial DNA after the PCR and determining that the microorganism is present when amplified; Microbial detection method comprising a.

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