PCR (Polymerase Chain Reaction) primer and kit for specifically detecting fusobacterium nucleatum and application of PCR primer and kit

Abstract

The invention belongs to the technical field of specific detection of pathogenic bacteria, and particularly relates to a PCR primer and a kit for specific detection of fusobacterium nucleatum and application of the PCR primer and the kit, in the primer sequence, the sequence of a forward primer is shown as SEQ ID NO.1, the sequence of a reverse primer is shown as SEQ ID NO.2, the primer can perform specific amplification on the fusobacterium nucleatum, and the primer can perform specific amplification on the fusobacterium nucleatum. The detection efficiency is improved, and the detection cost is reduced; the invention also provides a PCR method, a used PCR primer and a reaction system, and the fusobacterium nucleatum can be specifically detected under the background of a complex community; the method has the advantages of low cost, high speed and high accuracy, can avoid the defects of low speed, high cost, long period and the like of traditional experiments based on isolated culture of microorganisms, sequencing or mass spectrum identification and the like, and can be widely applied to rapid detection of fusobacterium nucleatum.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a PCR primer and a kit for specifically detecting Fusobacterium nucleatum and an application thereof. Background Art

[0002] The human intestinal microbiota contains microbial communities composed of fungi, bacteria, viruses, and protozoa, among which microbe-microbe and microbe-host interactions are crucial to human health. The imbalance of these interactions, reflected in changes in composition and function, is closely related to the occurrence and development of the disease. Colorectal cancer (CRC) is a common cancer. Studies have found that various genetic and environmental factors contribute to the development of CRC, from abnormal crypts to tumors. In CRC, the most closely related pathogenic bacteria is Fusobacterium nucleatum, which can be detected in CRC tissues, and its abundance gradually increases from normal tissues to adenoma tissues to colorectal cancer. Fusobacterium nucleatum enriched in tumors is involved in all stages of CRC progression. Therefore, the detection of Fusobacterium nucleatum can be used as one of the means to assist in the diagnosis of colorectal cancer.

[0003] In the past few decades, with the advancement of microbiome technology and bioinformatics, people have developed various methods for detecting bacteria, among which molecular biology is the most widely used, such as matrix-assisted laser desorption / ionization time-of-flight massspectrometry (MALDI-TO F MS) technology, qPCR method, serological ELISA, dot hybridization, in situ hybridization and other identification. At present, the clinical detection of Fusobacterium nucleatum (such as bacteremia, colorectal cancer tissue, oral swabs, etc.) is mainly based on culture medium, morphological detection, biochemical reaction identification, which takes 3-7 days and requires an anaerobic environment, which severely limits its application.

[0004] In view of the above technical problems, the present invention provides a PCR primer for specific detection of Fusobacterium nucleatum, analyzes the conserved gene sequence of Fusobacterium nucleatum, designs specific primers, and adopts the technology of fluorescent quantitative PCR to achieve rapid and accurate detection and identification of bacteria, and the method does not rely on culture and does not require an anaerobic environment. The method has the advantages of low cost, high speed and high accuracy, and can avoid the disadvantages of traditional experiments based on separation and culture of microorganisms, and then sequencing or mass spectrometry identification, such as slow speed, high cost and long cycle, and can be widely used in the rapid detection of Fusobacterium nucleatum. Summary of the invention

[0005] The object of the present invention is to provide a PCR primer for specifically detecting Fusobacterium nucleatum. The sequence of the forward primer in the primer sequence is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO.2.

[0006] The second object of the present invention is to provide the use of the PCR primers in detecting Fusobacterium nucleatum, wherein the detection is for non-disease diagnosis purposes.

[0007] The third object of the present invention is to provide the use of the PCR primers in preparing a kit for detecting Fusobacterium nucleatum.

[0008] The fourth object of the present invention is to provide a PCR kit for specifically detecting Fusobacterium nucleatum, wherein the kit comprises the PCR primers.

[0009] The fifth object of the present invention is to provide the use of the PCR kit in detecting Fusobacterium nucleatum, wherein the detection is for non-disease diagnosis purposes.

[0010] The sixth object of the present invention is to provide a PCR method for specifically detecting Fusobacterium nucleatum, extracting DNA of the microorganism to be detected, taking the DNA solution as a reaction template, and performing a PCR reaction with the primer combination.

[0011] Preferably, the reaction system in the method described is: 5 μL of 2×SYBR Green qPCR Master, 1 μL of template, 0.3 μL of upstream primer, 0.3 μL of downstream primer, and 3.4 μL of water.

[0012] Preferably, the PCR reaction program in the method described is: pre-denaturation at 95°C, 30s; denaturation / annealing / extension 40 cycles at 95°C, 10s, 95°C, 30s; melting curve at 95°C, 15s, 60°C, 60s, 95°C, 15s.

[0013] The beneficial effects of the present invention are:

[0014] (1) The present invention provides a PCR primer for specifically detecting Fusobacterium nucleatum, wherein the primer can specifically amplify Fusobacterium nucleatum, thereby improving detection efficiency and reducing detection costs;

[0015] (2) The present invention provides a PCR method for specifically detecting Fusobacterium nucleatum. The PCR primers and reaction system used can specifically detect Fusobacterium nucleatum in a complex community background.

[0016] (3) The method described has the advantages of low cost, high speed and high accuracy. It can avoid the disadvantages of traditional experiments based on isolation and cultivation of microorganisms and then sequencing or mass spectrometry identification, such as slow speed, high cost and long cycle. It can be widely used in the rapid detection of Fusobacterium nucleatum. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Phylogenetic analysis of Fusobacterium nucleatum ;

[0018] Figure 2 Conservative sequence analysis of Fusobacterium nucleatum;

[0019] Figure 3 Specificity verification of primers 1 to 5 for Fusobacterium nucleatum;

[0020] Figure 4 Standard curve of amplification efficiency and amplification range of primers 1 to 5 for Fusobacterium nucleatum;

[0021] Figure 5 Detection of Fusobacterium nucleatum in mixed communities;

[0022] Figure 6 Amplification curve of primer 1 of Fusobacterium nucleatum in mixed bacterial solution;

[0023] Figure 7 Detection of Fusobacterium nucleatum in stool samples and amplification curve using primer 1. DETAILED DESCRIPTION

[0024] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available unless otherwise specified.

[0025] Reagents and consumables: agar powder (Beijing Coolbo Technology Co., Ltd.), Gram staining kit (Beijing Solebow Technology Co., Ltd.), primer synthesis (Shanghai Biotechnology Co., Ltd.), GAM medium (HB8518-1, Qingdao Haibo Biological, 10.0 g / L of phenoxyethanol, 3.0 g / L of soytone, 5.0 g / L of yeast extract powder, 2.2 g / L of beef powder, 13.5 g / L of digested serum powder, 1.2 g / L of beef liver extract powder, 3.0 g / L of glucose, 2.5 g / L of potassium dihydrogen phosphate, 3.0 g / L of sodium chloride, 5.0 g / L of soluble starch, 0.3 g / L of L-cysteine, 0.3 g / L of sodium thioglycolate, 5 mg / ml of hemin chloride, 0.1% vitamin K1), anaerobic box (Mitsubishi MGC Co., Ltd., Japan), anaerobic bag (Mitsubishi MGC Co., Ltd., Japan), qPC R MIX (Wuhan Saiweier Biotechnology Co., Ltd.), bacterial genome extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), fecal genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), Fusobacterium nucleatum (Fn, China General Microbiological Culture Collection Administration Center, CGMCC 1.2526), ​​Bifidobacterium animalis (Bb, China General Microbiological Culture Collection Administration Center, CGMCC 1.15623), Limosilactobacillus reuteri (Institute of Microbiology, Guangdong Academy of Sciences, Lr, GDMCC 1.614), Akkermansia muciniphila (Institute of Microbiology, Guangdong Academy of Sciences, Ak, GDMCC 1.1346), Bacteroides thetaiotaomicron (Institute of Microbiology, Guangdong Academy of Sciences, Bt, GDMCC 1.1104), Clostridium sporogenes (Institute of Microbiology, Guangdong Academy of Sciences, Cs, GDMCC 1.1481), Parabacteroides distasonis (Institute of Microbiology, Guangdong Academy of Sciences, Pd, GDMCC 1.1564), Bacteroides fragilis (Institute of Microbiology, Guangdong Academy of Sciences, Bf, GDMCC 1.385).

[0026] Instruments: clean bench (Shanghai Shen'an Medical Instrument Factory), centrifuge (Hunan Hengnuo Instrument Equipment Co., Ltd.), vertical high-pressure steam sterilizer (Shanghai Shen'an Medical Instrument Factory), 680-type microplate reader (Bio-Rad, USA), constant temperature incubator (Tianjin Test Instrument Co., Ltd.), low-speed refrigerated centrifuge L530R (Xiangyi Centrifuge Instrument Co., Ltd.), NANODROP 2000 (Thermo Fisher Scientific), high-speed refrigerated centrifuge (Xiangyi Centrifuge Instrument Co., Ltd.), real-time quantitative PCR instrument (ABIQ5) (Thermo Fisher Scientific), data analysis software (R Software, MEGA Software, DNAman Software).

[0027] Example 1: Rapid Detection of Fusobacterium nucleatum

[0028] In this study, we systematically analyzed the developmental lineage of Fusobacterium nucleatum to determine the differences between its family members and other species. We downloaded the conserved gene sequence (16S gene) for multiple sequence alignment, and designed and synthesized specific primers based on the conserved sequence fragments using primer 3. We determined the amplification specificity by qPCR melting curve, established a conversion standard curve using Fusobacterium nucleatum clone counts and Ct values, and calculated the amplification efficiency based on the slope. Fusobacterium nucleatum was mixed cultured with a variety of other bacteria, and total DNA was extracted and tested to determine the detection performance of this method in a mixed background. Fecal DNA samples from clinical colorectal cancer patients were collected to determine the detection performance of this method in a complex background.

[0029] 1. Phylogenetic analysis of the Fusobacterium nucleatum family

[0030] (1) Download the FASTA files of the complete genomes of Fusobacterium nucleatum and Lactobacillus plantarum from NCBI, which contain the complete genome sequences of bacteria.

[0031] (2) Use barrnap to extract 16S rRNA sequences and save them in FASTA format.

[0032] (3) Use mega 11 software to construct a phylogenetic tree for the extracted 16S sequences.

[0033] The results are as follows Figure 1 As shown, Fusobacterium nucleatum has multiple subspecies, and phylogenetic analysis with Lactobacillus plantarum as the outgroup showed obvious distinction, indicating that the 16S sequence-based strategy can achieve accurate detection of Fusobacterium nucleatum.

[0034] 2. Analysis of Conserved Sequences of Fusobacterium nucleatum

[0035] (1) The 16S sequence of Fusobacterium nucleatum was imported into DNAman software and the consensus sequence was searched using the multiple sequence alignment function; the results were as follows: Figure 2 As shown, various Fusobacterium nucleatum have subspecies-specific sequence fragments as well as conserved common fragments. The common sequence is shown in SEQ ID NO.11, which provides a basis for primer design.

[0036] (2) The consensus sequence was partially input into the Primer BLAST module of NCBI to design specific primers. Primer pairs with amplification lengths of 150-250 bp were selected. A total of 5 pairs of primers were designed. The specific information is as follows:

[0037] Primer 1

[0038] Forward primer 5′-GGATTTATTGGGCGTAAAGC-3′ (SEQ ID NO. 1)

[0039] Reverse primer 5′-GGCATTCCTACAAATATCTACGAA-3′ (SEQ ID NO. 2)

[0040] Primer 2

[0041] Forward primer 5′-CGCAGAAGGTGAAAGTCCTGTAT-3′ (SEQ ID NO. 3)

[0042] Reverse primer 5′-TGGTCCTCACTGATTCACACAGA-3′ (SEQ ID NO. 4)

[0043] Primer 3

[0044] Forward primer 5′-CTAAATACGTGCCAGCAGC-3′ (SEQ ID NO. 5)

[0045] Reverse primer 5′-CGACCCCCAACACCTAGTA-3′ (SEQ ID NO. 6)

[0046] Primer 4

[0047] Forward primer 5′-CAACCATTACTTTAACTCTACCATGTTCA-3′ (SEQ ID NO. 7)

[0048] Reverse primer 5′-GTTGACTTTACAGAAGGAGATTATGTAAAAATC-3′ (SEQ ID NO. 8)

[0049] Primer 5

[0050] Forward primer 5′-GGATTTATTGGGCGTAAAGC-3′ (SEQ ID NO.9)

[0051] Reverse primer 5′-TACTGAGGGAGATTATGTAAAAATC-3′ (SEQ ID NO. 10)

[0052] 3. Validation of primer specificity for Fusobacterium nucleatum

[0053] (1) Synthesize 5 pairs of primers according to the primer sequences designed above;

[0054] (2) Follow the instructions for qPCR sample mixing and instrument detection (pre-denaturation at 95°C for 30 s; denaturation / annealing / extension for 40 cycles at 95°C for 10 s and 95°C for 30 s).

[0055] Dissolve 2×SYBR Green qPCR Master, template, primers and RNase-Free ddH2O, balance all reagents at room temperature and mix thoroughly. Prepare the reaction system according to (2×SYBR Green qPCR Master 5μL, template 1μL, upstream primer 0.3μL, downstream primer 0.3μL, water 3.4μL), cover the reaction tube, mix well, and centrifuge briefly. Place the reaction system in the qPCR instrument, set the reaction program according to (pre-denaturation 95℃, 30s; denaturation / annealing / extension 40 cycles, 95℃, 10s, 95℃, 30s; melting curve, 95℃, 15s, 60℃, 60s, 95℃, 15s), and start the reaction.

[0056] The results are as follows Figure 3 As shown, the melting curves of the five primer pairs were all single-peaked, and their specificity was qualified.

[0057] 4. Determination of the amplification efficiency and detection range of Fusobacterium nucleatum

[0058] (1) Fusobacterium nucleatum was cultured in GAM medium for 24 hours, centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and the suspension was resuspended in GAM medium, adjusted to OD = 1.0, and a 10-fold gradient dilution was performed from 10 -1 to 10 -8 , plate the plates and count the colonies (CFU).

[0059] (2) Take 1 ml of Fusobacterium nucleatum with OD=1.0 to extract bacterial DNA, perform gradient dilution, and amplify using qPCR to establish a standard curve between Ct value and CFU.

[0060] (3) According to the amplification efficiency = 10 (-1 / 斜率) *100% is used to calculate the amplification efficiency of this primer.

[0061] The results are as follows Figure 4 As shown, primer 1 has the best amplification efficiency, the linearity of the standard curve reaches 0.999, and the amplification efficiency is 92.03%. Therefore, primer 1 was used for subsequent experiments.

[0062] 5. Detection of Fusobacterium nucleatum in mixed community background

[0063] (1) Fusobacterium nucleatum (Fn) (China General Microbiological Culture Collection Center, CGMCC 1.2526), ​​Bifidobacterium animalis (Bb) (China General Microbiological Culture Collection Center, CGMCC 1.15623), Limosilactobacillus reuteri (Lr) (Institute of Microbiology, Guangdong Academy of Sciences, GDMCC 1.614), Akkermansia muciniphila (Ak) (Institute of Microbiology, Guangdong Academy of Sciences, GDMCC 1.1346), Bacteroides thetaiotaomicron (Bt) (Institute of Microbiology, Guangdong Academy of Sciences, GDMCC 1.1104), Clostridium sporogenes (Cs) (Institute of Microbiology, Guangdong Academy of Sciences, GDMCC 1.1481), Parabacteroides distachyon (Bt) (Institute of Microbiology, Guangdong Academy of Sciences, GDMCC 1.1626), and Bacteroides spp. distasonis, Pd) (Institute of Microbiology, Guangdong Academy of Sciences, GDMCC 1.1564), Bacteroides fragilis (Bf) (Institute of Microbiology, Guangdong Academy of Sciences, GDMCC 1.385), 8 bacteria were cultured in GAM medium for 24-48 hours, centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and the solution was resuspended in GAM medium, the OD was adjusted to 1.0, 1 ml of the bacterial solution was taken, the genomic DNA was extracted, and the concentration was determined.

[0064] (2) Perform qPCR sample mixing and instrument detection according to the instructions (pre-denaturation at 95°C for 30 s; denaturation / annealing / extension for 40 cycles at 95°C for 10 s and 95°C for 30 s).

[0065] Dissolve 2×SYBR Green qPCRMaster, template, primers and RNase-Free ddH2O, balance all reagents at room temperature and mix thoroughly. Prepare the reaction system according to (2×SYBR Green qPCRMaster 5μL, template 1μL, upstream primer 0.3μL, downstream primer 0.3μL, water 3.4μL), cover the reaction tube, mix well, and centrifuge briefly. Place the reaction system in the qPCR instrument, set the reaction program according to (pre-denaturation 95℃, 30s; denaturation / annealing / extension 40 cycles, 95℃, 10s, 95℃, 30s; melting curve, 95℃, 15s, 60℃, 60s, 95℃, 15s), and start the reaction.

[0066] Lr:

[0067] 5'-GGAGAAGAACGTGCGTGAGA-3'

[0068] 5'-AGCCGAAGGCTTTCACATCA-3'

[0069] Bt:

[0070] 5'-GAGGAAGGTCCCCCACATTG-3'

[0071] 5'-ACCCATAGGGCAGTCATCCT-3'

[0072] Ak:

[0073] 5'-CAGCACGTGAAGGTGGGGAC-3'

[0074] 5'-CCTTGCGGTTGGCTTCAGAT-3'

[0075] Cs:

[0076] 5'-TTGGCTCTGCACCGGGAATC-3'

[0077] 5'-CTGCAAACGCCGTCCCTCTT-3'

[0078] Pd:

[0079] 5'-CGGTTTCGTGCGGGTGATGA-3'

[0080] 5'-CGGAAGGTGGCGTTCGTGTT-3'

[0081] Bf:

[0082] 5'-CACTTGACTGTTGTAGATAAAGC-3'

[0083] 5'-CATCTTCATTGCAGCATTATCC-3'

[0084] Bb:

[0085] 5'-CACCAATGCGGAAGACCAG-3'

[0086] 5'-GTTGTTGAGAATCAGCGTGG-3'

[0087] The results are as follows Figure 5 As shown in Figure 2, all eight bacteria were specifically amplified. Figure 6 As shown, primer 1 and the reaction system can specifically detect Fusobacterium nucleatum in a complex community background, and the amplification efficiency is qualified (92.17%).

[0088] 6. Detection of Fusobacterium nucleatum in fecal background

[0089] (1) Stool sample collection: Prepare a sterile stool collection cup, collect about 0.5 g of stool from colorectal cancer patients, and immediately store it at -80°C.

[0090] (2) Use a DNA extraction kit to extract genomic DNA from the sample: Weigh 180-220 mg of fecal sample into a 2 ml centrifuge tube and place the tube on ice. Add 500 μl of buffer SA, 100 μl of buffer SC, 15 μl of Proteinase K, and 0.25 g of grinding beads to the sample and oscillate intermittently for 1 min until the sample is fully mixed. Incubate at 70°C for 15 min, oscillating 2-3 times during incubation. Vortex for 15 s, centrifuge at 12,000 rpm for 3 min, transfer the supernatant to a new centrifuge tube, add 10 μl of RNaseA, oscillate to mix, and place at room temperature for 5 min. Add 200 μl of buffer SH, oscillate to mix, and place on ice for 5 min. Centrifuge at 12,000 rpm for 3 min. Transfer the supernatant obtained in the previous step to a new 1.5 ml centrifuge tube and add an equal volume of buffer GFA. Add the solution obtained in the previous step to an adsorption column CR2 (the adsorption column is placed in a collection tube), centrifuge at 12.000rpm for 30s, discard the waste liquid, and place the adsorption column CR2 in a collection tube. Add 500μl of buffer GD to the adsorption column CR2, centrifuge at 12000rpm for 30s, discard the waste liquid, and place the adsorption column CR2 in a collection tube. Add 700μl of rinse solution PW to the adsorption column CR2, centrifuge at 12000rpm for 30s, discard the waste liquid, and place the adsorption column CR2 in a collection tube. Repeat the above cleaning steps. Put the adsorption column CR2 back into the collection tube, centrifuge at 12000rpm for 2min, and discard the waste liquid. Leave the adsorption column CR2 at room temperature for a few minutes to thoroughly dry the residual rinse solution in the adsorption material. Transfer the adsorption column CR2 into a clean centrifuge tube, add 100 μl of elution buffer TB to the middle of the adsorption membrane, place it at room temperature for 5 minutes, centrifuge it at 12000 rpm for 2 minutes, and collect the solution into a centrifuge tube. Then use agarose gel electrophoresis and NanoDrop2000 to detect the concentration of DNA.

[0091] (3) Perform qPCR sample mixing and instrument detection according to the instructions (pre-denaturation at 95°C for 30 s; denaturation / annealing / extension for 40 cycles at 95°C for 10 s and 95°C for 30 s).

[0092] Dissolve 2×SYBR Green qPCR Master, template, primers and RNase-Free ddH2O, balance all reagents at room temperature and mix thoroughly. Prepare the reaction system according to (2×SYBR Green qPCR Master 5μL, template 1μL, upstream primer 0.3μL, downstream primer 0.3μL, water 3.4μL), cover the reaction tube, mix well, and centrifuge briefly. Place the reaction system in the qPCR instrument, set the reaction program according to (pre-denaturation 95℃, 30s; denaturation / annealing / extension 40 cycles, 95℃, 10s, 95℃, 30s; melting curve, 95℃, 15s, 60℃, 60s, 95℃, 15s), and start the reaction.

[0093] The results are as follows Figure 7 As shown, in the complex background of feces, the reaction system can still maintain excellent amplification specificity, and the amplification efficiency reaches 95.75%.

[0094] In summary, the present invention provides a PCR primer for specific detection of Fusobacterium nucleatum, wherein the primer can specifically amplify Fusobacterium nucleatum, thereby improving detection efficiency and reducing detection cost;

[0095] The present invention provides a PCR method for specifically detecting Fusobacterium nucleatum, and the PCR primers and reaction system used can specifically detect Fusobacterium nucleatum in a complex community background. The method has the advantages of low cost, high speed and high accuracy, and can avoid the disadvantages of traditional experiments based on separation and cultivation of microorganisms, and then sequencing or mass spectrometry identification, such as slow speed, high cost and long cycle, and can be widely used in the rapid detection of Fusobacterium nucleatum.

[0096] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A PCR primer for specific detection of Fusobacterium nucleatum, characterized in that: The sequence of the forward primer in the primer sequence is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO.

2.

2. Use of the PCR primers as claimed in claim 1 in detecting Fusobacterium nucleatum, wherein the detection is for non-disease diagnosis purposes.

3. Use of the PCR primers according to claim 1 in preparing a kit for detecting Fusobacterium nucleatum.

4. A PCR kit for specific detection of Fusobacterium nucleatum, characterized in that: The kit comprises the PCR primers according to claim 1.

5. Use of the PCR kit as claimed in claim 4 in detecting Fusobacterium nucleatum, wherein the detection is for non-disease diagnosis purposes.

6. A PCR method for specifically detecting Fusobacterium nucleatum, characterized in that: Extract the DNA of the microorganism to be tested, take the DNA solution as a reaction template, and carry out PCR reaction with the primer combination described in claim 1.

7. The PCR method according to claim 6, characterized in that The reaction system in the method described is: 2×SYBR Green qPCR Master 5 μL, template 1 μL, upstream primer 0.3 μL, downstream primer 0.3 μL, water 3.4 μL.

8. The PCR method according to claim 6, characterized in that The PCR reaction program in the method described is: pre-denaturation at 95°C for 30 s; denaturation / annealing / extension for 40 cycles at 95°C for 10 s, 95°C for 30 s; melting curve at 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s.

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