CN110592184A - Method for extracting and identifying Brucella genome DNA - Google Patents

Abstract

The invention discloses a method for extracting and identifying brucella genome DNA. The method comprises the following steps: the method comprises the steps of separating pure cultured brucella from blood cells of blood of patients with diseases, extracting genome DNA of the pure cultured brucella, and extracting brucella DNA from serum of the patients. The invention provides a novel method for separating, pure culturing and extracting brucella DNA from patient whole blood, which is simple, convenient, rapid and accurate to operate, is suitable for large-scale blood examination and provides a novel technology for brucella infectious disease diagnosis.

Description

Method for extracting and identifying Brucella genome DNA

Technical Field

The invention relates to the fields of medicine and biotechnology. Specifically, the invention relates to an extraction and identification method of infectious pathogen brucella genome DNA.

Background

Brucella (Brucella) disease, also known as malachite fever or wave fever, is abbreviated as Brucella. Is one of the most serious zoonosis in the world, and has the occurrence and the prevalence in 170 countries and regions. Of the 76 diseases investigated which endangered human health, the Buffalo disease was the seventh. Official statistics of over 50 million people worldwide are infected with the disease 1 each year. Because brucellosis seriously affects the development of animal husbandry and brings great threat to human health, the prevention, treatment and detection of brucellosis are increasingly paid more attention by governments of various countries.

The brucellosis is specified as a second type of infectious disease by the animal epidemic prevention law in China, and the epidemic of the brucellosis is controlled once in the beginning of 90 years in the 20 th century after years of efforts. However, with the rapid development of livestock husbandry in China in recent years, the market circulation of cattle and sheep is frequent, the breeding scale is enlarged, and the incidence rate of brucellosis of technical personnel of livestock veterinarians in pasture areas and grassroots livestock veterinarians tends to increase. According to statistics of Chinese disease prevention centers, the number of the diseases infected with brucellosis in China per year is nearly 5 ten thousand in average since 2010. Provinces and autonomous regions such as inner Mongolia, Gansu, Qinghai, Ningxia, Xinjiang, Hebei, Shanxi, Heilongjiang, Jilin, Liaoning and the like in China are main regions for animal husbandry production in China, and patients with Brucella are found in people and livestock.

The occurrence, development and prognosis of brucellosis are complex, the clinical manifestations are various, and the diagnosis of one symptom is difficult to determine. The diagnosis of brucellosis should be comprehensively judged by combining the epidemiological contact history, clinical manifestations, laboratory examinations and other conditions of patients. For example, a patient before the disease is attacked has a history of close contact with livestock and livestock products suspected to be infected by the Brucella, or has eaten the milk and meat products of cattle and sheep, or lives in a Brucella disease affected area; or the culture and detection of the Brucella or the production and use of the Brucella vaccine. The clinical manifestations are as follows: fever (including low fever), hyperhidrosis, fatigue, muscle and joint pain, etc. lasting for days or even weeks. The lymph nodes, liver, spleen and testis of some patients are swollen, and a small number of patients can have various rashes and jaundice; patients in acute and chronic stages, manifested by damage to the osteoarticular system.

Traditionally, the gold standard for diagnosing brucellosis is isolated culture of brucella, which is high in risk, long in time (14 ~ 40 days), high in laboratory hardware requirement (laboratories above grade P2), and almost ineffective in chronic brucellosis.

Serological methods are currently the most commonly used detection method for cloth sickness. Since the body can produce specific antibodies about one week after infection with brucella, serological diagnostic techniques can diagnose brucellosis by detecting the antibodies. The serological diagnostic techniques commonly used mainly include test tube agglutination tests (SAT), tiger red plate agglutination tests (RBPT), antiglobulin tests (AHG or Coombs tests), Complement Fixation Tests (CFT), enzyme-linked immunosorbent assay (ELISA), and other detection methods. Among them, the tiger red plate agglutination test (RBPT) is widely used domestically because of its low cost, and the enzyme-linked immunosorbent assay (ELISA) is specified as a detection method in international trade by OIE because of its high specificity and high sensitivity. However, serological methods fail to detect the presence of brucella when antibodies are not produced early in the infection.

The molecular biological method can specifically detect brucella nucleic acid, can improve the accuracy of brucella clinical diagnosis, has long time of the fluorescent quantitative PCR detection method, and needs expensive equipment and professional operators. The traditional PCR method is relatively simple and convenient to operate, and the common PCR instrument is more widely popularized and has better popularization. However, no commercial reagent has been approved for SFDA to date.

Whether a fluorescence quantitative PCR detection method or a traditional PCR detection method is adopted, in order to improve the detection sensitivity and accuracy, the problem of extracting nucleic acid from brucella is solved firstly, the separation and extraction of brucella DNA from brucella is a key point of detection, particularly, when a patient is infected with brucella in the initial stage, the content of pathogenic bacteria in blood of the patient is low, nucleic acid is extracted through a series of steps, the finally obtained brucella DNA is very small in amount, sometimes, DNA cannot be extracted, a false negative result appears by adopting fluorescence quantitative PCR or traditional PCR detection, and the timely treatment of the patient is delayed.

In particular, to date, there is no mature complete nucleic acid extraction protocol or technique available for brucella.

Disclosure of Invention

The invention aims to provide a method for extracting and identifying brucella genome DNA, so as to solve the technical problem.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for extracting and identifying Brucella genome DNA is characterized by comprising the following steps:

(1) isolating cultured monocytes from the patient's blood;

(2) separating brucella from the mononuclear cells and extracting genomic DNA;

(3) extracting brucella DNA from patient serum;

(4) measuring the content and purity of brucella DNA separated and extracted from blood cells and serum;

(5) carrying out agarose gel electrophoresis identification on the extracted brucella DNA;

(6) designing PCR primers of the brucella abortus specific and conservative gene, and carrying out PCR amplification on the extracted brucella abortus DNA to obtain the characteristic gene so as to confirm that the brucella abortus DNA is extracted from the serum and blood cells of a patient.

As a further scheme of the present invention, the step (1) specifically comprises the following steps:

1) taking 1ml of human peripheral blood; adding 1ml Solution I (1640 culture Solution), mixing, spreading on 6-hole cell culture plate, and incubating for 2 hr at 37 deg.C;

2) washing with 1-2ml Solution II (0.1 mM PBS buffer Solution with pH 7.2), washing to remove erythrocytes in the culture plate, washing repeatedly for 2-3 times, and removing erythrocytes to obtain pure monocyte.

As a further scheme of the present invention, the step (2) specifically comprises the following steps:

1) adding 200 μ L Solution III (50 mmol/L Tris-Cl, 50mmol/LEDTA, 0.5% Tween-20, 0.5% Triton X-100, pH8.0), suspending the thallus, mixing well, adding 10 μ L protein kinase K (20 mg/mL), 5 μ L lysozyme (100 mg/mL) and 2 μ L NaseA (20 mg/mL), mixing well, incubating at 37 deg.C for at least 30min, heating to 65 deg.C, incubating for 10min, or mixing well until the Solution is clear;

2) adding 200 μ L of anhydrous ethanol, mixing by inversion (the solution may be turbid), adding 30 μ L of isopropanol into lysate (containing any precipitate), mixing, centrifuging at 13000Xg for 1min, and removing supernatant;

3) adding 500 μ L of solution 2 (1.25M NaCl, 20mM Tris-Cl, 15% isopropanol, pH 7.0), centrifuging at 13000Xg for 1min, and discarding the supernatant;

4) adding 500 μ L of solution 3 (70% ethanol TE solution), centrifuging at 13000Xg for 1min, and discarding the supernatant; the process can be repeated once;

5) 50 ~ 100 μ L of TE buffer (10 mM/L of Tirs-Cl, 1mmol/L of EDTA, pH 8.0) or DNase-free water was added, the mixture was allowed to stand at 65 ℃ for 5min, and 13000Xg was centrifuged for 1min, and the solution was collected and stored at-20 ℃.

As a further scheme of the present invention, the step (3) specifically comprises the following steps:

1) serum was centrifuged at 8000Xg for 5min at 200. mu.L, and the supernatant was discarded. Washing the suspended cells with 200. mu.L of solution 1 (50 mmol/L Tris-Cl, 50mmol/LEDTA, 0.5% Tween-20, 0.5% Triton X-100, pH 8.0), adding 10. mu.L protein kinase K (20 mg/mL), 5. mu.L lysozyme (100 mg/mL) and 2. mu.L LRNaseA (20 mg/mL), and mixing; incubating at 37 deg.C for at least 30min, heating to 65 deg.C, incubating for 10min, and mixing to clear solution;

2) adding 200 μ L of anhydrous ethanol, mixing by inversion (the solution may be turbid), adding 30 μ L of isopropanol, and mixing;

3) transferring the liquid (including the possible precipitate) to an adsorption column, and centrifuging at 13000Xg for 1 min;

4) add 500. mu.L of solution 2 (1.25M NaCl, 20mM Tris-Cl, 15% isopropanol, pH 7.0), centrifuge at 13000Xg for 1 min;

5) adding 500 μ L of solution 3 (70% ethanol TE solution), centrifuging at 13000Xg for 1min, and repeating the steps once;

6) centrifuging for 2min by an empty tube 13000 Xg; adding 30-50 μ L TE buffer (10 mM/L Tirs-Cl, 1mmol/L EDTA, pH 8.0) or DNase-free H2O into the center of the adsorption column, and standing at room temperature for 5 min;

7) 13000Xg for 1min, collect the solution and store at-20 ℃.

As a further scheme of the present invention, the step (4) specifically comprises the following steps:

the extracted genome is identified by Nanodrop, and the concentrations of the extracted genome from brucella serum and blood cells are 141 ng/muL and 133 ng/muL respectively, and A260/A280 is 1.82 and 1.86 respectively, which are seen from Table 1, and indicate that the extracted genome DNA has higher concentration and no protein and RNA pollution;

sample (I)	Bacterial type	Concentration ng/. mu.L	A260	A280	260/280
						Blood serum	Sheep type 1	141	2.82	1.55	1.82
Blood cells	Sheep type 1	133	2.66	1.43	1.86

Table 1 brucella genomic DNA concentration versus purity table.

As a further scheme of the present invention, the step (5) specifically comprises the following steps: preparing 1% agarose gel by TBS (butyl phosphate buffer) with pH8.0, identifying brucella DNA extracted from mononuclear cells and brucella in serum, carrying out electrophoresis by TBS with pH8.0, carrying out electrophoresis voltage of 80mV and electrophoresis time of 30 minutes in electrophoresis buffer solution of 0.5M, dyeing by EB, and observing electrophoresis results under a gel imaging system.

As a further scheme of the present invention, the step (6) specifically comprises the following steps:

identifying BCSP31 gene with the identification gene being periplasmic protein, identifying the designed upstream primer BCSP 31-F: 5' -GCGcggatcCAAATTCGGAAGCAAAATCCG-3', and a downstream primer BCSP 31-R: 5' -CGGctcgagTTATTTCAGCACGCCCGC-3`；

The conditions for PCR amplification were: 50ul of reaction system, 0.25 mu mol/L of each of BCSP31-F and BCSP31-R, 0.1 mu g of template DNA, 200 mu mol/L of 4XdNTP, 2.5u of Tag DNA polymerase, and the conditions of PCR reaction are as follows: denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30 sec, and extension at 72 ℃ for 60 sec for a total of 30 cycles.

Compared with the prior art, the invention has the following advantages: the invention cultures and separates the monocyte from the patient's blood, separate Brucella in monocyte and extract Brucella DNA from it, extract Brucella DNA from patient's serum; performing quality evaluation on brucella DNA separated and extracted from blood cells and serum; the extracted brucella DNA is subjected to PCR amplification by designing a specific PCR primer of the brucella conservative gene so as to confirm that the brucella DNA is extracted from serum and blood cells of a patient. The invention breaks the bottleneck of brucella nucleic acid extraction, and wins precious time for accurately diagnosing whether the patient is infected with brucella by adopting conventional PCR and fluorescent quantitative PCR, preventing the patient from being transferred into chronic infection, striving for advanced treatment and early recovery of the patient.

Drawings

FIG. 1 is a diagram showing the purity of Brucella DNA separated and extracted from blood cells and serum according to the present invention by Nanodrop;

FIG. 2 shows that the periplasmic protein BCSP31 of the invention is a target gene, and a specific upstream primer BCSP31-F is designed: 5' -GCGcggatcCAAATTCGGAAGCAAAATCCG-3' and a downstream primer BCSP 31-R: 5' -CGGctcgagTTATTTCAGCACGCCCGC-3' by PCR amplification, and determining whether the cloth is extracted by the above methodGenomic DNA profile of L.reuteri (wherein A: from blood sample and B: from serum sample.).

Detailed Description

The invention is explained in further detail below with reference to the figures and the specific embodiments.

1) Isolating cultured monocytes from the patient's blood:

taking 1ml of human peripheral blood; adding 1ml of 1640 culture solution, mixing uniformly, and incubating for 2 hours at 37 ℃ on a cell culture plate; the mononuclear cells were obtained by washing with 1 to 2ml of 0.1mM PBS buffer pH 7.2 and washing the red blood cells in the plate 2 to 3 times.

2) Isolating brucella from the monocytes and extracting the genomic DNA:

(1) add 200. mu.L Solution III (50 mmol/L Tris-Cl, 50mmol/LEDTA, 0.5% Tween-20, 0.5% Triton X-100, pH 8.0), suspend the cells, mix well, add 10. mu.L protein kinase K (20 mg/mL), 5. mu.L lysozyme (100 mg/mL) and 2. mu.L LRNaseA (20 mg/mL), mix well. Incubate at 37 ℃ for at least 30 min. Raising the temperature to 65 ℃, incubating for 10min, and mixing the solution for a while until the solution is clear;

(2) add 200. mu.L of absolute ethanol and mix by inversion. (the solution may be turbid), adding 30. mu.L of isopropanol into the lysate (including any precipitate), mixing, centrifuging at 13000Xg for 1min, and discarding the supernatant;

(3) adding 500 μ L of solution 2 (1.25M NaCl, 20mM Tris-Cl, 15% isopropanol, pH 7.0), centrifuging at 13000Xg for 1min, and discarding the supernatant;

(4) adding 500 μ L of solution 3 (70% ethanol TE solution), centrifuging at 13000Xg for 1min, and discarding the supernatant; the process can be repeated once;

(5) 50 ~ 100 μ L of TE buffer (10 mM/L of Tirs-Cl, 1mmol/L of EDTA, pH 8.0) or DNase-free water was added, the mixture was allowed to stand at 65 ℃ for 5min, and 13000Xg was centrifuged for 1min, and the solution was collected and stored at-20 ℃.

3) Extracting brucella DNA from the serum of a patient:

(1) serum was centrifuged at 8000Xg for 5min at 200. mu.L, and the supernatant was discarded. The suspended cells were washed with 200. mu.L of solution 1 (50 mmol/L Tris-Cl, 50mmol/LEDTA, 0.5% Tween-20, 0.5% Triton X-100, pH 8.0). Adding 10 μ L of protein kinase K (20 mg/mL), 5 μ L of lysozyme (100 mg/mL) and 2 μ L of LRNaseA (20 mg/mL), and mixing; incubate at 37 ℃ for at least 30 min. Raising the temperature to 65 ℃, incubating for 10min, and mixing the solution for a while until the solution is clear;

(2) adding 200 μ L of anhydrous ethanol, mixing by inversion (the solution may be turbid), adding 30 μ L of isopropanol, and mixing;

(3) transferring the liquid (including the possible precipitate) to an adsorption column, and centrifuging at 13000Xg for 1 min;

(4) add 500. mu.L of solution 2 (1.25M NaCl, 20mM Tris-Cl, 15% isopropanol, pH 7.0), centrifuge at 13000Xg for 1 min;

(5) adding 500 μ L of solution 3 (70% ethanol TE solution), centrifuging at 13000Xg for 1min, and repeating the steps once;

(6) centrifuging for 2min by an empty tube 13000 Xg; 30-50. mu.L of TE buffer (10 mM/L of Tirs-Cl, 1mmol/L of EDTA, pH 8.0) or DNase-free H2O was added to the center of the column and allowed to stand at room temperature for 5 min.

(7) 13000Xg for 1min, collect the solution and store at-20 ℃.

4) And measuring the content and purity of the brucella DNA separated and extracted from the blood cells and the serum:

the concentration and purity of genomic DNA extracted from brucella serum and blood cells was tested by Nanodrop. As shown in FIG. 1, the ratios of Brucella DNA 260/280 isolated from blood and serum are shown to be 1.86 and 1.82, respectively.

5) And carrying out agarose gel electrophoresis identification on the extracted brucella DNA:

the brucella DNA extracted from the mononuclear cells and the serum is identified by agarose gel electrophoresis and EB staining, and the electrophoresis result is observed under a gel imaging system.

6) Designing a PCR primer of the brucella abortus specificity and conservative gene, and carrying out PCR amplification on the extracted brucella abortus DNA to obtain the characteristic gene so as to confirm that the brucella abortus DNA is extracted from the serum and blood cells of a patient.

Identification of genes as BCSP31 genes of periplasmic proteinsTherefore, the designed upstream primer BCSP31-F is identified: 5' -GCGcggatcCAAATTCGGAAGCAAAATCCG-3', and a downstream primer BCSP 31-R: 5' -CGGctcgagTTATTTCAGCACGCCCGC-3'; the conditions for PCR amplification were: 50ul of reaction system, 0.25 mu mol/L of BCSP31-F and BCSP31-R respectively, 0.1 mu g of template DNA and 200 mu mol/L of 4 XdNTP. The amount of Tag DNA polymerase was 2.5 u. The conditions of the PCR reaction were: denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30 sec, and extension at 72 ℃ for 60 sec for a total of 30 cycles. As shown in fig. 2, the periplasmic protein BCSP31 was targeted, and a specific upstream primer BCSP31-F was designed: 5' -GCGcggatcCAAATTCGGAAGCAAAATCCG-3' and a downstream primer BCSP 31-R: 5' -CGGctcgagTTATTTCAGCACGCCCGC-3' and identifying whether the DNA extracted by the method is the genome DNA of the Brucella through PCR amplification. Experimental results prove that the DN band of the amplification product is 1006bp, and is consistent with the expected size, thereby proving that the method for extracting the genome DNA of the brucella is correct. A: from blood samples, B from serum samples.

The foregoing is a preferred embodiment of the present invention, and it will be apparent to those skilled in the art that variations, modifications, substitutions and alterations can be made in the embodiment without departing from the principles and spirit of the invention.

Claims (7)

1. A method for extracting and identifying Brucella genome DNA is characterized by comprising the following steps:

(1) isolating cultured monocytes from the patient's blood;

(2) separating brucella from the mononuclear cells and extracting genomic DNA;

(3) extracting brucella DNA from patient serum;

(4) measuring the content and purity of brucella DNA separated and extracted from blood cells and serum;

(5) carrying out agarose gel electrophoresis identification on the extracted brucella DNA;

(6) designing PCR primers of the brucella abortus specific and conservative gene, and carrying out PCR amplification on the extracted brucella abortus DNA to obtain the characteristic gene so as to confirm that the brucella abortus DNA is extracted from the serum and blood cells of a patient.

2. The method for extracting and identifying brucella genomic DNA according to claim 1, wherein the step (1) specifically comprises the following steps:

1) taking 1ml of human peripheral blood; adding 1ml Solution I (1640 culture Solution), mixing, spreading on 6-hole cell culture plate, and incubating for 2 hr at 37 deg.C;

2) washing with 1-2ml Solution II (0.1 mM PBS buffer Solution with pH 7.2), washing to remove erythrocytes in the culture plate, washing repeatedly for 2-3 times, and removing erythrocytes to obtain pure monocyte.

3. The method for extracting and identifying brucella genomic DNA according to claim 1, wherein the step (2) specifically comprises the following steps:

1) adding 200 μ L Solution III (50 mmol/L Tris-Cl, 50mmol/LEDTA, 0.5% Tween-20, 0.5% Triton X-100, pH8.0), suspending the thallus, mixing well, adding 10 μ L protein kinase K (20 mg/mL), 5 μ L lysozyme (100 mg/mL) and 2 μ L NaseA (20 mg/mL), mixing well, incubating at 37 deg.C for at least 30min, heating to 65 deg.C, incubating for 10min, or mixing well until the Solution is clear;

2) adding 200 μ L of anhydrous ethanol, mixing by inversion (the solution may be turbid), adding 30 μ L of isopropanol into lysate (containing any precipitate), mixing, centrifuging at 13000Xg for 1min, and removing supernatant;

3) adding 500 μ L of solution 2 (1.25M NaCl, 20mM Tris-Cl, 15% isopropanol, pH 7.0), centrifuging at 13000Xg for 1min, and discarding the supernatant;

4) adding 500 μ L of solution 3 (70% ethanol TE solution), centrifuging at 13000Xg for 1min, and discarding the supernatant; the process can be repeated once;

5) 50 ~ 100 μ L of TE buffer (10 mM/L of Tirs-Cl, 1mmol/L of EDTA, pH 8.0) or DNase-free water was added, the mixture was allowed to stand at 65 ℃ for 5min, and 13000Xg was centrifuged for 1min, and the solution was collected and stored at-20 ℃.

4. The method for extracting and identifying brucella genomic DNA according to claim 1, wherein the step (3) specifically comprises the following steps:

1) centrifuging serum 200 μ L at 8000Xg for 5min, discarding supernatant, washing suspended thallus with 200 μ L solution 1 (50 mmol/L Tris-Cl, 50mmol/LEDTA, 0.5% Tween-20, 0.5% Triton X-100, pH8.0), adding 10 μ L protein kinase K (20 mg/mL), 5 μ L lysozyme (100 mg/mL) and 2 μ L NaseA (20 mg/mL), and mixing; incubating at 37 deg.C for at least 30min, heating to 65 deg.C, incubating for 10min, and mixing to clear solution;

2) adding 200 μ L of anhydrous ethanol, mixing by inversion (the solution may be turbid), adding 30 μ L of isopropanol, and mixing;

3) transferring the liquid (including the possible precipitate) to an adsorption column, and centrifuging at 13000Xg for 1 min;

4) add 500. mu.L of solution 2 (1.25M NaCl, 20mM Tris-Cl, 15% isopropanol, pH 7.0), centrifuge at 13000Xg for 1 min;

5) adding 500 μ L of solution 3 (70% ethanol TE solution), centrifuging at 13000Xg for 1min, and repeating the steps once;

6) centrifuging for 2min by an empty tube 13000 Xg; adding 30-50 μ L TE buffer (10 mM/L Tirs-Cl, 1mmol/L EDTA, pH 8.0) or DNase-free H2O into the center of the adsorption column, and standing at room temperature for 5 min;

7) 13000Xg for 1min, collect the solution and store at-20 ℃.

5. The method for extracting and identifying brucella genomic DNA according to claim 1, wherein the step (4) specifically comprises the following steps:

the extracted genome is identified by Nanodrop, and the concentrations of the extracted genome from brucella serum and blood cells are 141 ng/muL and 133 ng/muL respectively, and A260/A280 is 1.82 and 1.86 respectively, which are seen from Table 1, and indicate that the extracted genome DNA has higher concentration and no protein and RNA pollution;

sample (I) Bacterial type Concentration ng/. mu.L A260 A280 260/280 Blood serum Sheep type 1 141 2.82 1.55 1.82 Blood cells Sheep type 1 133 2.66 1.43 1.86

Table 1 brucella genomic DNA concentration versus purity table.

6. The method for extracting and identifying brucella genomic DNA according to claim 1, wherein the step (5) specifically comprises the following steps: preparing 1% agarose gel by TBS (butyl phosphate buffer) with pH8.0, identifying brucella DNA extracted from mononuclear cells and brucella in serum, carrying out electrophoresis by TBS with pH8.0, carrying out electrophoresis voltage of 80mV and electrophoresis time of 30 minutes in electrophoresis buffer solution of 0.5M, dyeing by EB, and observing electrophoresis results under a gel imaging system.

7. The method for extracting and identifying brucella genomic DNA according to claim 1, wherein the step (6) specifically comprises the following steps:

identifying BCSP31 gene with the identification gene being periplasmic protein, identifying the designed upstream primer BCSP 31-F: 5' -GCGcggatcCAAATTCGGAAGCAAAATCCG-3', and a downstream primer BCSP 31-R: 5' -CGGctcgagTTATTTCAGCACGCCCGC-3`；

The conditions for PCR amplification were: 50ul of reaction system, 0.25 mu mol/L of each of BCSP31-F and BCSP31-R, 0.1 mu g of template DNA, 200 mu mol/L of 4XdNTP, 2.5u of Tag DNA polymerase, and the conditions of PCR reaction are as follows: denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30 sec, and extension at 72 ℃ for 60 sec for a total of 30 cycles.