WO2020199342A1 - 常温核酸扩增反应 - Google Patents

常温核酸扩增反应 Download PDF

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WO2020199342A1
WO2020199342A1 PCT/CN2019/090586 CN2019090586W WO2020199342A1 WO 2020199342 A1 WO2020199342 A1 WO 2020199342A1 CN 2019090586 W CN2019090586 W CN 2019090586W WO 2020199342 A1 WO2020199342 A1 WO 2020199342A1
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protein
temperature
nhis
sequence
amplification
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PCT/CN2019/090586
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French (fr)
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于继彬
李俊
马陈翠
高山珊
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苏州先达基因科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1223—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/03—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
    • C12Y207/03002—Creatine kinase (2.7.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to an enzyme with low-temperature activity and its application to carry out nucleic acid amplification reactions under low-temperature in vitro conditions.
  • PCR Polymerase chain reaction
  • RT- PCR Reverse Transcript, reverse transcription PCR
  • qPCR Quantantitative PCR, fluorescent quantitative PCR
  • nested PCR etc.
  • the PCR reaction requires cleavage of DNA double-strands into single strands under high temperature conditions, and then cooling and annealing the primers to pair with the template strands, and finally the primers are extended at 72°C.
  • the above process repeats the DNA exponential amplification.
  • the PCR reaction process needs to be carried out in an instrument with precise temperature control components.
  • Such instruments are often expensive and require very complex professional skills for operators. Therefore, they are only equipped in laboratories or medical institutions in some developed regions. This greatly limits the promotion and application of PCR-based molecular diagnostic technology.
  • LAMP and RPA are the most widely used, but LAMP and RPA still need to be reacted at a specific temperature (LAMP: 60-65°C, RPA: 37-42°C), and their reaction is more sensitive to temperature changes. If the temperature is not the most suitable temperature, the amplification efficiency will be greatly reduced or the amplification will not be correct.
  • T4 phage replication-related proteins (gene protein 32, 41, 43, 44, 62, 45, and 61) Realize the high-efficiency replication of double-stranded DNA in vitro, and the extension speed of DNA can almost reach the amplification efficiency of 500 bases/sec in vivo [2].
  • the gp32 protein plays a key role in the DNA replication, recombination and repair of T4 phage. The most important of these is that the gp32 protein has the characteristics of tightly binding single-stranded DNA [3]. In 1983, Formosa et al.
  • gp32 protein immobilized on an agarose adsorption column to perform affinity chromatography on the bacterial fusion products infected by T4 phage, and found that the DNA polymerase (gp43 protein) in T4 phage and two important recombination pathway proteins (uvsX and uvsY proteins) can specifically bind to gp32 protein [4].
  • the uvsX protein was confirmed in subsequent studies that it has a similar function to recA in Escherichia coli, has DNA-dependent ATPase activity, and can bind to single-stranded or double-stranded DNA under physiological saline conditions in vitro, and catalyze it with the same Source double-stranded or single-stranded DNA fragments are paired [5,6]. In the same period, Deborah et al.
  • uvsX catalyzes the hydrolysis of ATP at a rate of 10-20 times that of recA, and the catalytic products can be AMP+PP i and ADP+P i . Hydrolysis of ATP is more complete, while recA catalyzes ATP to produce ADP+P i [7].
  • the gp32 protein can greatly stimulate the activity of uvsX to catalyze the homologous pairing of single-stranded DNA (ssDNA).
  • ssDNA single-stranded DNA
  • uvsX can bind tightly to uvsY [7]
  • uvsY can increase the single-stranded DNA dependence of uvsX by enhancing the affinity between uvsX and ssDNA.
  • ATPase activity [8, 9].
  • the homologous recombination function carried out by uvsX combined with ssDNA is closely related to the replication process of T4 DNA. After uvsX-ssDNA is combined with homologous fragments, ssDNA can be used as a primer for DNA replication [10].
  • UvsY and Gp32-primer complexes are formed UvsY-Gp32-primer complex, and reduce the binding affinity of Gp32 and primer, so that UvsX competitively binds to the primer to form UvsX-primer complex;
  • UvsX-primer complex has a template under the action of ATP The characteristic of homologous pairing at complementary sites in the chain, and replace the chain with the same base sequence, and then combine with the complementary chain to form a D-loop structure;
  • the exposed 3'end of the primer in the D-loop structure is in the DNA polymerase
  • the primer is extended under the action of, until the replication is completed.
  • the target DNA fragments can be amplified exponentially by reciprocating the above process.
  • this patent uses Gp32, UvsX and UvsY, DNA polymerase, creatine kinase and other proteins to perform in vitro Combination screening of low-temperature phage protein or amino acid mutation screening allows the amplification reaction to be efficiently amplified at an exponential rate at a lower temperature and shorten the amplification time.
  • the reaction can even completely separate from the temperature control equipment, and only need the general indoor ambient temperature for rapid amplification; combined with the nucleic acid detection method, the entire nucleic acid amplification and detection equipment required is simplified and the operation is more convenient; the reaction can be selected The range of media is more extensive, and the amplification reaction can be performed not only on heated media, such as fiber paper, nylon membrane, nitrocellulose membrane, cotton fiber ball, etc.
  • T4-related phage hosts About 90% of the known T4-related phage hosts are Escherichia coli or other enteric bacilli, and the remaining 10% are hosts of other bacteria, such as Aeromonas, Vibrio or Synechococcus, etc. [23]. Most of them are found in domestic sewage or wastewater. The natural host is human or other animal intestines[24]. Therefore, their optimal growth temperature is similar to their host, 37-40 degrees[25]. According to them The optimal culture temperature for plaque formation is usually divided into three categories: high temperature (HT) phage above 25 degrees, low temperature (LT) phage below 30 degrees, and normal temperature for plaque formation between 15-42 degrees. Sex (MT) phage[26]. T4 phage is a typical room temperature phage.
  • the optimum temperature of these three phage is determined to be 24 degrees Celsius.
  • the protein sequence derived from the low-temperature phage is significantly different from the T4 phage.
  • the vB_EcoM-VR5 phage uvsX protein has only 65.6% homology with T4.
  • the corresponding DNA sequence was synthesized by referring to the above-mentioned genome sequence, and cloned into pET22b expression vector with Nde I and EcoR I respectively, and the expressed proteins were named VR5X_NHis, VR7_25X_NHis, VR20_26X_NHis, VR5G_NHis, VR7G_NHis, VR25G_NHis, VR7Y_NHis, VR5Y_NHis, VR5Y_NHis, respectively VR20Y_NHis, VR7_25X_CHis, VR7G_CHis, VR7_25_26Y_CHis, the sequence is as follows:
  • T4 phage corresponding genes uvsX, uvsY and gp32 were synthesized and cloned into the expression vector pET22b through molecular biology.
  • the expressed amino acid sequence is as follows:
  • host cell BL21 (DE3) was transformed, IPTG induced expression, repeated freezing and thawing and purification by Ni column [28], and the high purity protein was obtained and then amplified and tested.
  • UvsX functions similar to E. coli RecA protein, and its homologous strand displacement transferase activity depends on ATP. Unlike RecA, uvsX decomposes ATP into two products, ADP and AMP. High concentrations of ADP and AMP can inhibit uvsX [31], so the product needs to be converted into ATP to reduce the inhibition.
  • ATP concentration is 1mM-5mM, creatine phosphate and muscle kinase [32, 33].
  • Myokinase can choose rabbit myokinase, carp myokinase.
  • NP_001075708.1 protein sequence was synthesized and its genes were synthesized and cloned into pET22b expression vector with Nde I and EcoR I respectively.
  • the corresponding proteins were named RM-CK and Carp-M1- CK, and fused 6xHis tag at the N-terminus to facilitate purification.
  • the RM-CK encoding gene was mutated, so that the translated protein amino acid 268 (without histidine tag) G was mutated to N.
  • the amplification reaction conditions refer to the Sinha, N.K. method, the Mg2+ ion concentration is 5-20mM, the K+ ion concentration is 20-120mM, preferably, the concentration is 40-80mM; the dNTP concentration is 100uM-1000uM, preferably, between 300uM-600uM.
  • room temperature amplification reaction system is constructed as follows:
  • the reaction conditions are as follows: 25ul, amplification temperature: 20-45°C. Use water bath, constant temperature equipment or PCR machine. The end of the reaction is monitored by agarose gel electrophoresis, Sybr Green I or specific probes. During Sybr green I monitoring, the final concentration of the reaction system is increased to 0.3-0.5 x Sybr green I. The reaction time can be 20-40min, and the fluorescence is read every 30s. Fluorescence channel: FAM/HEX. The detection instrument can use ABI7500, FTC-3000, Bio-Rad CFX MiniOpticon System, GenDx constant temperature fluorescence detector GS8, etc.
  • Probe labeling refers to [22] for design and synthesis. Fluorescence detection adopts ABI7500, FTC-3000, Bio-Rad CFX MiniOpticon System, GenDx constant temperature fluorescence detector GS8, etc.
  • the beneficial effects of the present invention applying the low-temperature phage protein provided by the present invention to a normal temperature nucleic acid amplification reaction can not only realize nucleic acid amplification and detection at a lower temperature, but also further improve the sensitivity of detection, and can detect 100 copies /ul nucleic acid.
  • Figure 1 shows the test VR5, VR7, VR20 and T4 (control) of spot formation rate at different temperatures.
  • Figure 2 shows the evolutionary tree analysis of Geneious v5.5 genome.
  • Figure 3 shows the sequence comparison of uvsX between different species.
  • Figure 4 is a curve diagram of constant temperature amplification using Enterobacteria phage vB_EcoM_VR5 amplification system.
  • Figure 5 is a curve diagram of constant temperature amplification using Enterobacteria phage vB_EcoM_VR7 amplification system.
  • Figure 6 is a curve diagram of constant temperature amplification using a mixed type protein amplification system derived from different species.
  • Figure 7 is the electrophoresis diagram of the amplified products obtained by low-temperature amplification using Enterobacteria phage vB_EcoM_VR5 amplification system and RPA (Recombinase polymerase amplification) technology.
  • Bands 1, 2, and 3 are respectively using RPA amplification reagent (TALQBAS01) at 20°C Amplification results under conditions; bands 4, 5, and 6 were amplified using RPA amplification reagent (TALQBAS01) at 25°C; bands 7, 8, and 9 were amplified using Enterobacteria phage vB_EcoM_VR5 phage protein at 20°C, respectively Amplification results under conditions; bands 10, 11, and 12 are the results of amplification using Enterobacteria phage vB_EcoM_VR5 phage protein at 25°C.
  • Figure 8 is a curve diagram of constant temperature amplification using mutant creatine kinase and wild-type creatine kinase amplification systems.
  • Figure 9 is a graph of constant temperature amplification curves using different polymerases in the reaction system.
  • Figure 10 is a graph of sensitivity detection.
  • Figure 11 is a graph showing the constant temperature amplification curve of the reaction system using different uvsX mutants.
  • Figure 12 shows the effect of different temperatures on the amplification efficiency of the amplification system using low-temperature phage proteins.
  • Figure 13 shows the constant temperature amplification curve of VRX_Variant1 450ng/ul; VR5G_NHis 550ng/ul; VR5Y_NHis 60ng/ul amplification system for sample detection.
  • Example 1 Construction of recombinant protein expression vector and protein expression and purification
  • the gene sequence corresponds to the C-terminus of the protein fused with 6 histidine tags, which is named gene + CHis, and the protein N-terminus Fusion of 6 histidine tags is named gene +NHis.
  • gene + CHis the protein fused with 6 histidine tags
  • gene +NHis the protein N-terminus Fusion of 6 histidine tags
  • the gene number is added at the same time, such as VR7_25_26Y_CHis, which means that the uvsY amino acid sequence of the three strains of vB_EcoM_VR7, vB_EcoM_VR25 and vB_EcoM_VR26 are consistent, and this number corresponds to
  • the protein has a 6XHis tag added to the C-terminus.
  • the reaction reagents and their concentrations are as follows: Tris-acetic acid buffer, 30mM; potassium acetate, 60mM; magnesium acetate 20mM; dithiothreitol 2mM; polyethylene glycol (molecular weight 1450-20000) 5%; ATP, 3mM; creatine phosphate, 30mM; creatine kinase, 90ng/ul; VR5X_NHis protein, 200-600ng/ul; VR5G_NHis protein, 200-1000ng/ul; VR5Y_NHis protein, 60ng/ul; Staphylococcus aureus polymerase I large fragment (exo-), 8Units; dNTP, 450uM; upstream primer, 250nM; downstream primer, 250nM; template is Mycoplasma pneumoniae genomic DNA template about 10ng/ul; Sybr Green I final concentration, 0.4x .
  • VR5G_NHis protein 1000ng/ul VR5X_NHis protein, 300ng/ul
  • VR5G_NHis protein 200ng/ul VR5X_NHis protein, 600ng/ul
  • Enterobacteria phage vB_EcoM_VR7 amplification system was constructed, and the effect of different terminal His tags on protein activity was tested.
  • the reagents and their concentrations are as follows: Tris-acetic acid buffer, 100mM; potassium acetate, 120mM; magnesium acetate 15mM; dithiothreitol 6mM; polyethylene glycol (molecular weight 1450-20000) 6%; ATP , 2mM; creatine phosphate, 40mM; creatine kinase, 75ng/ul; VR7_25X_NHis or VR7_25X_CHis protein, 400ng/ul; VR7G_NHis or VR7G_CHis protein, 480ng/ul; VR7_25_26Y_NHis or VR7_25_26Y_CHis protein, 80ng/ul; Bacillus subtilis polymerase Large fragment (exo-), 8Units; dNTP, 450uM; exo exonuclease
  • ARMP-R 5’-CCATGCACCATCTGTCACTCCGTTAACCTCCG-3’ (SEQ ID NO.42)
  • ARMP-PB 5’-TGTTACGCGGAGAACCTTACCCAC(Fam-dT)(THF)T(BHQ1-dT)GACATCCTTCGCAAT-3’(SEQ ID NO.43)
  • reaction conditions are as follows: 50ul, amplification temperature: 32°C.
  • the amplification results are as follows.
  • VR7_25X_NHis protein 400ng/ul
  • VR7G_NHis protein 480ng/ul
  • VR7_25_26Y_NHis protein 80ng/ul.
  • VR7_25X_NHis protein 400ng/ul
  • VR7G_CHis protein 480ng/ul
  • VR7_25_26Y_NHis protein 80ng/ul
  • VR7_25X_CHis protein 400ng/ul
  • VR7G_CHis protein 480ng/ul
  • VR7_25_26Y_NHis protein 80ng/ul
  • the detection threshold of the amplified fluorescent signal (the reaction time that can monitor the change of the fluorescent signal value, TT, Threshold Time) are basically the same. It proved that under the same protein concentration, the His protein tag at the N-terminus or C-terminus of the fusion protein has no significant difference in the effect of protein activity.
  • Tris-acetic acid buffer 50mM; potassium acetate, 80mM; magnesium acetate 20mM; dithiothreitol 2mM; polyethylene glycol (molecular weight 1450-20000) 6%; ATP , 2mM; creatine phosphate, 30mM; creatine kinase, 60ng/ul; VR5X_NHis, VR7_25X_NHis or VR20_26X_NHis protein, all 400ng/ul; VR7G_NHis or VR25G_NHis protein, 600ng/ul; VR7_25_26Y_NHis or VR20Y_NHis protein, golden yellow Staphylococcal polymerase I large fragment (exo-), 8Units; dNTP, 450uM; exo exonuclease III, 50ng/ul; upstream primer sus
  • the primer and probe sequences are as follows:
  • S1/S3/S5/S7 are templates for adding genomic DNA.
  • S2/S4/S6/S8 are NTC negative controls.
  • S1/S3/S5/S7 are NTC negative controls.
  • S2/S4/S6/S8 are templates for adding genomic DNA.
  • Example 5 Using Enterobacteria phage vB_EcoM_VR5 amplification system to test the effect of different temperatures on amplification efficiency
  • Enterobacteria phage vB_EcoM_VR5 amplification reaction reagents and their concentrations are as follows: Tris-acetic acid buffer, 20mM; potassium acetate, 120mM; magnesium acetate 10mM; dithiothreitol 8mM; polyethylene glycol (molecular weight 20000)5 %; ATP, 3mM; creatine phosphate, 20mM; creatine kinase, 30ng/ul; VR5X_NHis protein, 350ng/ul; VR5G_NHis protein, 500ng/ul; VR5Y_NHis protein, 50ng/ul; Bacillus subtilis polymerase I large fragment ( exo-), 10Units; dNTP, 450uM; upstream primer peu-F: 5'-GCGAACGGGTGAGTAACACGTATCCAATCT-3' (SEQ ID NO.47), 250nM; downstream primer peu-R1: 5'-AGCCATTACCTGCTAAAGTCATTCTTCCCAAA
  • TwistDx reagent www.twistdx.co.uk product catalog number: TALQBAS01
  • TwistDx reagent www.twistdx.co.uk product catalog number: TALQBAS01
  • Three replicates were set for each trial. Use a water bath to control the reaction temperature. After reacting for 1 hour, after the protein is inactivated by a high temperature of 80 degrees immediately, the amplified product is recovered by alcohol precipitation, and then dissolved in 20ul TE, and 10ul of the recovered product is detected by gel electrophoresis. Increase the result. , As shown in Figure 7.
  • the 16srDNA segment gene sequence carrying Mycoplasma pneumoniae is:
  • the sequence was cloned into the EcoR V restriction blunt-end site on the pUC57 vector.
  • the amplification efficiency of Enterobacteria phage vB_EcoM_VR5 phage-derived protein is significantly better than that of T4 phage-derived at low temperature.
  • the reagents and their concentrations are as follows: Tris-acetic acid buffer, 30mM; potassium acetate, 60mM; magnesium acetate 8mM; dithiothreitol 4mM; polyethylene glycol (molecular weight 1450-20000) 3%; ATP , 3mM; creatine phosphate, 50mM; RM-CK/RM-CK_G268N/Carp-M1-CK, 30-50ng/ul; VR7_25X_NHis protein, 360ng/ul; VR7G_NHis protein, 500ng/ul; VR7_25_26Y_NHis protein, 60ng/ul; Bacillus subtilis polymerase I large fragment (exo-), 8Units; dNTP, 450uM; upstream primer susF, 250nM; downstream primer susR, 250nM; template is the total genomic DNA template of pork tissue about 10ng/ul; detected by probe susPB, nfo endonuclease IV, the final concentration is 130ng/ul
  • the reaction conditions are as follows: 25ul, amplification temperature: 32°C.
  • the reaction was carried out on the GS8 fluorescence amplification instrument of Xianda Gene, the fluorescence scan interval was 60S, and the reaction time was 40min. The result is shown in Figure 8.
  • Example 7 The influence of different polymerases on amplification efficiency
  • the reaction system is: VR7_25X_NHis protein, 300ng/ul; VR7G_CHis protein, 400ng/ul; VR7_25_26Y_NHis protein, 50ng/ul, polymerase using Staphylococcus aureus polymerase I large fragment (exo-)/Bacillus subtilis polymerase I large fragment (exo-)/E. coli klenow polymerase large fragment (exo-)/Pseudomonas fluorescens polymerase I large fragment (exo-) are all 100ng/ul, other reagents and their concentrations are the same as in Example 5, with additional additions Sybr Green I 0.4X, amplification temperature: 33°C.
  • the reaction was carried out on the GS8 fluorescence amplification instrument of Xianda Gene, the fluorescence scanning interval was 30S, and the reaction time was 20min. The amplification results are shown in Figure 9.
  • S1/S2 Staphylococcus aureus polymerase I large fragment (exo-)
  • S1/S3/S5/S7 are templates for adding genomic DNA.
  • S2/S4/S6/S8 are NTC negative controls.
  • the amplification efficiency of the other three DNA polymerases is relatively high except that the amplification efficiency of E. coli klenow polymerase large fragment (exo-) is slightly lower.
  • the reagents and their concentrations are as follows: Tris-acetic acid buffer, 50mM; potassium acetate, 100mM; magnesium acetate 16mM; dithiothreitol 2mM; polyethylene glycol (molecular weight 1450-20000) 6%; ATP , 2.5mM; creatine phosphate, 30mM; creatine kinase, 120ng/ul; VR7_25X_NHis protein, 450ng/ul; VR7G_NHis protein, 700ng/ul; VR7_25_26Y_NHis protein, 70ng/ul; Staphylococcus aureus polymerase I large fragment (exo -), 8Units; dNTP, 450uM; upstream primer, 250nM; downstream primer, 250nM; template is the plasmid sequence synthesized by grass carp reovirus GCRV VP7 protein gene, respectively diluted to 10,000,000 copies/ul, 1,000,000 copies/ul, 100,000 copies /ul, 10000 copies/ul,
  • reaction conditions are as follows: 50ul, amplification temperature: 35°C.
  • GCRV-I-R313 5’-TCCAATTCGTGATAGTCTACAGTACGGCTACC-3’ (SEQ ID NO.55)
  • the gene sequence of the GCRV VP7 protein gene carrying the grass carp reovirus is:
  • the sequence was cloned into the EcoR V restriction blunt-end site on the pUC57 vector.
  • the reaction was carried out on the GS8 fluorescence amplification instrument of Xianda Gene, the fluorescence scanning interval was 30S, and the reaction time was 20min.
  • test results shown in Figure 10 show that the amplification of S6 samples is very obvious, for S7 samples, there is a slight increase in fluorescence signal. Therefore, the detection sensitivity can be no less than 100 copies/ul, which is close to the detection sensitivity of other molecular diagnostic techniques. By optimizing primer and probe sequences, it should be expected to obtain better results to achieve the detection of a single copy of the amplified fluorescent signal.
  • the reagents and their concentrations are as follows: Tris-acetic acid buffer, 20mM; potassium acetate, 120mM; magnesium acetate, 10mM; polyethylene glycol (molecular weight 1450-20000) 6%; ATP, 4mM; creatine phosphate, 45mM; Creatine Kinase, 90ng/ul; Twenty different mutant uvsX proteins, 450ng/ul; VR7G_CHis protein, 550ng/ul; VR7_25_26Y_NHis protein, 60ng/ul; Staphylococcus aureus polymerase I large fragment (exo-) , 120ng/ul; dNTP, 450uM; upstream primer ARMP-F, 400nM; downstream primer ARMP-R, 400nM; template is a plasmid template carrying a 16srDNA gene sequence of Mycoplasma pneumoniae about 3000 copies/ul; Sybr Green I concentration is 0.5 X;
  • the reaction conditions are as follows: 50ul,
  • VRX_Variant1 450ng/ul; VR5G_NHis 550ng/ul; VR5Y_NHis 60ng/ul;
  • VRX_Variant2 450ng/ul; VR5G_NHis 550ng/ul; VR5Y_NHis 60ng/ul;
  • VRX_Variant3 450ng/ul; VR5G_NHis 550ng/ul; VR5Y_NHis 60ng/ul;
  • VRX_Variant4 450ng/ul; VR5G_NHis 550ng/ul; VR5Y_NHis 60ng/ul;
  • NTC VR5X_NHis 450ng/ul; VR5G_NHis 550ng/ul; VR5Y_NHis 60ng/ul; no template.
  • VRX_Variant8 450ng/ul; VR7G_NHis 550ng/ul; VR7_25_26Y_NHis 60ng/ul;
  • NTC VR7_25X_NHis 450ng/ul; VR7G_NHis 550ng/ul; VR7_25_26Y_NHis 60ng/ul; no template.
  • NTC VR20_26X_NHis 450ng/ul; VR25G_NHis 550ng/ul; VR20Y_NHis 60ng/ul; no template.
  • NTC VR7_25X_NHis 450ng/ul; VR7G_NHis 550ng/ul; VR7_25_26Y_NHis 60ng/ul; no template.
  • NTC VR20_26X_NHis 450ng/ul; VR25G_NHis 550ng/ul; VR7_25_26Y_NHis 60ng/ul; no template.
  • the reagents and their concentrations are as follows: Tris-acetic acid buffer, 30mM; potassium acetate, 60mM; magnesium acetate 8mM; dithiothreitol 4mM; polyethylene glycol (molecular weight: 20000) 5%; ATP, 3mM ; Creatine phosphate, 50mM; RM-CK, 30ng/ul; VR7_25X_NHis protein, 360ng/ul; VR7G_NHis protein, 500ng/ul; VR7_25_26Y_NHis protein, 60ng/ul; Bacillus subtilis polymerase I large fragment (exo-), 8Units ; DNTP, 450uM; upstream primer susF, 250nM; downstream primer susR, 250nM; template is pork tissue total genomic DNA template about 10ng/ul; using probe detection, the final concentration of probe susPB is 120nM, exonuclease III (exo III), the final concentration is 70ng/ul.
  • the reaction conditions are as follows: 50ul; in addition, using RPA technology, using TwistDx reagent (www.twistdx.co.uk catalog number: TALQBAS01), and increasing the final concentration to 70ng/ul exonuclease III (exo III) as a comparison, other expansions
  • Amplification temperature 20-45°C, with a temperature gradient every five degrees.
  • the amplification results proved that, unlike the RPA amplification reagent, the low-temperature protein system derived from VR7 has a more obvious amplification effect at 20-30 degrees Celsius, while the RPA amplification reagent has higher amplification efficiency at 35-40 degrees. Consistent with reports in the literature, in this experiment, under the condition of 20 degrees Celsius, the RPA reagent did not detect the change of the amplified fluorescence signal value.
  • Example 11 Using VRX_Variant1, VR5G_NHis, VR5Y_NHis protein combination amplification to detect whether there is mycoplasma contamination in a cell sample
  • Tris-acetate buffer 100mM; potassium acetate, 120mM; magnesium acetate 15mM; dithiothreitol 6mM; polyethylene glycol (molecular weight: 20000) 5%; ATP, 2mM ; Creatine phosphate, 40mM; Creatine kinase, 75ng/ul; VRX_Variant1 450ng/ul; VR5G_NHis 550ng/ul; VR5Y_NHis 60ng/ul; Bacillus subtilis polymerase I large fragment (exo-), 8Units; dNTP, 450uM; Sybr Green I 0.4X.
  • Upstream primer ARMP-F 250nM; downstream primer ARMP-R, 250nM; fluorescent probe ARMP-PB, 120nM; primer and probe sequences are:
  • ARMP-F 5’-AGCATGTGGTTTAATTTGATGTTACGCGG-3’ (SEQ ID NO.59)
  • ARMP-R 5’-CCATGCACCATCTGTCACTCCGTTAACCTCCG-3’ (SEQ ID NO.60)
  • the reaction conditions are as follows: 50ul, amplification temperature: 32°C.
  • the sample is the cell culture fluid confirmed to be contaminated by mycoplasma; the fluorescence curve is detected after amplification.
  • Sample processing Take 500 ⁇ l of cell supernatant (or the above-mentioned cell suspension), centrifuge at 14000rpm for 6min, remove the supernatant to collect the precipitate (Note: Use a pipette to clean the supernatant), add 50 ⁇ l of sterile water, shake and mix, 95 After a water bath at °C for 3 minutes, shake and mix gently. After rapid centrifugation, the DNA template will be released into the supernatant. During the reaction, 2.5ul is added to the system as a template.

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Abstract

本发明提供了一种低温噬菌体蛋白在常温核酸扩增反应中的应用，其中低温噬菌体选自vB_EcoM-VR5,vB_EcoM-VR7and vB_EcoM-VR20,vB_EcoM-VR25,vB_EcoM-VR26，低温噬菌体蛋白为uvsX蛋白、uvsY蛋白和gp32蛋白和/或具有相应功能的突变体蛋白，优选情况下uvsX蛋白及其突变体蛋白选自SEQ ID No.1-23、30任一条序列；uvsY蛋白及其突变体蛋白选自SEQ ID No.27-29、32任一条序列；gp32蛋白及其突变体蛋白选自SEQ ID No.24-26、31任一条序列。本发明还提供了包含低温噬菌体蛋白的常温核酸扩增反应体系。

Description

[根据细则37.2由ISA制定的发明名称]　常温核酸扩增反应 技术领域
本发明属于生物技术领域，具体涉及一种具有低温活性的酶及其在低温体外条件下进行核酸扩增反应的应用。
背景技术
体外核酸扩增技术在现代生命科学领域中是一项及其重要的技术领域。自1990年以来，体外核酸扩增技术领域得到了飞速的发展，不仅如此，该技术领域还将在未来的生命科学领域中扮演越来越重要的作用。
人类和动物的很多疾病可以通过核酸技术进行早期诊断，而这依赖于一种有效的体外核酸扩增技术。聚合酶链式反应(Polymerase chain reaction,PCR)是一种经典的体外核酸扩增方法，从诞生到现在已经延用了30多年，并且基于PCR衍生出各种不同功能核酸检测方法，如RT-PCR(Reverse Transcript,反转录PCR)，qPCR(Quantitative PCR,荧光定量PCR)，巢式PCR等。PCR的反应需要在高温条件下将DNA双链裂解成单链，而后降温退火引物与模板链配对，最后在72℃条件下引物得到延伸，以上过程循环往复DNA呈指数式扩增。PCR的反应过程需要在具有精密温控元件的仪器中进行，这种仪器往往价格昂贵，同时对操作人员需要非常复杂的专业技能，因而只有在一些发达地区的实验室或者医疗机构才会配备，这大大限制了基于PCR的分子诊断技术推广应用。鉴于传统PCR受限于仪器设备和电力等因素的限制，近些年来恒温体外核酸扩增技术悄然兴起，如SDA(Strand displacement amplification,链置换扩增技术)，HDA(Helicase dependent amplification,解旋酶依赖性扩增技术)，NASBA(Nuclear acid sequence-based amplification，核酸依赖性扩增检测技术)，LAMP(Loop-mediated isothermal amplification,环介导等温扩增技术)，RCA(Rolling circle amplification,滚环复制扩增技术),RPA(Recombinase Polymerase Amplification,重组酶聚合酶扩增技术)等等[1]。其中应用最广泛的是LAMP和RPA，但是LAMP和RPA仍然需要在特定的温度下(LAMP：60-65℃，RPA：37-42℃)进行反应，而且其反应对温度的变化比较敏感，在非最适合温度下，其扩增效率会大大降低或者无法正确扩增。
早期人们对DNA的体内复制进行了深入的研究，其中因T4噬菌体结构简单，侵入细菌后会将其基因组DNA在胞内能进行快速的复制，前人将其作为模式生物进行了大量的病毒学和分子遗传学研究。1976年YASUO IMAE等人利用噬菌体裂解液蛋白进行了T4噬菌体DNA的体外复制[29]；1979年，Sinha等又利用T4噬菌体复制相关蛋白(基因蛋白32,41,43,44,62,45,and 61)实现了双链DNA的体外高效复制，DNA的延伸速度近乎可以达到与体内的500个碱基 /秒的扩增效率[2]。gp32蛋白在T4噬菌体的DNA复制、重组和修复过程中起着关键作用，这其中最重要的是因为gp32蛋白具有紧密结合单链DNA的特性[3]。1983年，Formosa等将gp32蛋白固定在琼脂糖吸附柱上对T4噬菌体感染的细菌融菌产物进行亲和层析，发现T4噬菌体中的DNA聚合酶(gp43蛋白)和两种重要的重组通路蛋白(uvsX和uvsY蛋白)可以和gp32蛋白进行特异性结合[4]。uvsX蛋白在之后的研究中证实，它与大肠杆菌中的recA具有相似的功能，具有DNA依赖性ATP酶活性，可以在体外生理盐水条件下与单链或双链DNA结合，并催化其与同源的双链或单链DNA片段进行配对[5,6]。同期，Deborah等通过克隆重组表达了uvsX基因，并验证了uvsX蛋白在gp32蛋白存在下，具有ATP依赖性将单链置换双链DNA的活性，从而形成D-Loop结构，并将底物ATP分解为ADP和AMP两种产物[7A]。与recA不同的是，uvsX催化水解ATP的速率是recA的10-20倍，而且催化产物可以是AMP+PP i和ADP+P i，水解ATP更彻底，而recA催化ATP只能产生ADP+P i[7]。gp32蛋白可以大大刺激uvsX催化单链DNA(ssDNA)同源配对的活性，同时uvsX又可以与uvsY紧密结合[7]，而且uvsY可以通过加强uvsX与ssDNA的亲和力来提高uvsX的单链DNA依赖性ATP酶活性[8，9]。由uvsX结合ssDNA后进行的同源重组功能与T4 DNA的复制过程紧密关联，uvsX-ssDNA与同源片段结合后，ssDNA可以作为一个引物进行DNA复制[10]。以上的证据表明，gp32、uvsX和uvsY蛋白在T4噬菌体DNA的复制过程中起到非常重要的作用。之后的研究中发现，uvsY与ssDNA形成的uvsY-ssDNA复合体可以引起ssDNA结构的改变，使ssDNA构架更加倾向于形成uvsX-SSDNA复合体，同时ATP的结合更加稳定了uvsX-ssDNA的结构[11-14]。进一步的研究发现，gp32蛋白与单链ssDNA形成稳定的gp32-ssDNA复合体，但是在uvsY的作用下，gp32-ssDNA复合体结构的稳定性明显减弱，同时使ssDNA更加倾向于形成uvsX-ssDNA复合体[15]。
David A.Zarling等人利用这一特征，通过大肠杆菌RecA蛋白和SSB蛋白，利用特异性引物进行了特定区段的核酸扩增；Piepenburg在此基础上又做了改进，并将大肠杆菌蛋白替换为T4噬菌体蛋白gp32、uvsX、uvsY，DNA聚合酶选用bsu DNA聚合酶大片段或sau DNA聚合酶大片段，并命名为RPA扩增(recombinase polymerase amplification)[22]。Gp32蛋白功能类似于细菌中的SSB蛋白，它可以特异性与ssDNA寡核苷酸引物，就形成Gp32-引物复合物，使引物保持单链结构；第二步，UvsY与Gp32-引物复合物形成UvsY-Gp32-引物复合物，并降低Gp32与引物结合的亲和力，从而使UvsX竞争性的与引物结合形成UvsX-引物复合物；第三步，UvsX-引物复合物在ATP的作用下具有与模板链中互补位点同源配对的特性，并置换出碱基序列相同的链，而后与互补链结合形成D-loop结构；第四步D-loop结构中引物暴露的3’端在DNA聚合酶的作用下引物得到延伸，直至复制完成。以上过程循环往复靶 DNA片段就可呈指数式扩增。
尽管据报道RPA技术可以在30度条件下能够扩增，但其扩增效率大大降低，为了进一步降低反应温度，本专利通过针对Gp32、UvsX和UvsY，DNA聚合酶，肌酸激酶等蛋白进行体外组合筛选低温噬菌体蛋白或进行氨基酸突变筛选，使扩增反应可以在更低的温度下以指数式速率高效扩增，缩短扩增时间。这样甚至可以彻底脱离温控设备，只需要一般室内环境温度即可进行快速扩增；结合核酸检测方法，使整个核酸扩增和检测所需的设备更加简化，操作更加便捷；对于反应的可以选用的介质范围更加广泛，扩增反应可以不局限于加热的介质上进行,如纤维纸片,尼龙膜,硝酸纤维素膜,棉纤维球等。
发明内容
大约90％已知的T4相关的噬菌体的宿主为大肠杆菌或其它肠道杆菌，其余10％的宿主为其它细菌类，如产气单胞菌，弧菌或聚球藻菌等[23].绝大多数被发现于生活污水或废水中，天然宿主为人类或其它动物肠道[24].因此它们的最适生长温度如他们的宿主类似，均为37-40度[25].根据它们形成噬菌斑的最适培养温度不同通常分成三类：25度以上称为高温型(HT)噬菌体，30度以下的低温型(LT)噬菌体，15-42度之间形成噬菌斑的常温性(MT)噬菌体[26].T4噬菌体是典型的常温噬菌体。
Laura Kaliniene等先后发现了五株低温噬菌体,vB_EcoM-VR5,vB_EcoM-VR7 and vB_EcoM-VR20,vB_EcoM-VR25,vB_EcoM-VR26。并根据Seeley and Primrose[27]方法在17,24,30,35,37,39 and 40C温度下分别对vB_EcoM-VR5,vB_EcoM-VR7和vB_EcoM-VR20三株噬菌体进行空斑试验，发现此三株对温度敏感性与T4噬菌体明显不同，其成斑率如图1所示，并确定此三株噬菌体最适温度为24摄氏度。通过对基因组进行测序(Enterobacteria phage vB_EcoM_VR5,Accession:KP007359.1；vB_EcoM-VR7,Accession:HM563683.1；vB_EcoM_VR20,Accession:KP007360.1；vB_EcoM_VR25,Accession:KP007361.1；vB_EcoM_VR26,Accession:KP007362.1)，并获得了其DNA复制相关的蛋白序列。
经alignX软件比较(如图3所示)，低温噬菌体来源的蛋白序列与T4噬菌体有明显不同，如vB_EcoM-VR5噬菌体uvsX蛋白与T4同源性只有65.6％。
参照上述基因组序列合成相应的DNA序列，并分别采用Nde I和EcoR I双酶切克隆至pET22b表达载体，其表达的蛋白分别命名为VR5X_NHis，VR7_25X_NHis，VR20_26X_NHis，VR5G_NHis，VR7G_NHis，VR25G_NHis，VR5Y_NHis，VR7_25_26Y_NHis，VR20Y_NHis，VR7_25X_CHis，VR7G_CHis，VR7_25_26Y_CHis,序列如下：
Figure PCTCN2019090586-appb-000001
Figure PCTCN2019090586-appb-000002
Figure PCTCN2019090586-appb-000003
Figure PCTCN2019090586-appb-000004
同时合成T4噬菌体对应的基因uvsX，uvsY和gp32，并通过分子生物学克隆至表达载体pET22b。其表达的氨基酸序列如下：
Figure PCTCN2019090586-appb-000005
Figure PCTCN2019090586-appb-000006
根据分子克隆实验指南技术进行转化宿主细胞BL21(DE3)，IPTG诱导表达，反复冻融裂解并采用Ni柱纯化[28]，获得高纯度蛋白后进行扩增测试。
UvsX功能为类似于大肠杆菌RecA蛋白，它的同源链置换转移酶活性依赖于ATP。不同于RecA，uvsX将ATP分解后成为ADP和AMP两种产物。高浓度的ADP和AMP会对uvsX产生抑制作用[31]，因此需要将产物转化为ATP以降低抑制。参考Hinton,D.M、
Figure PCTCN2019090586-appb-000007
等方法配制能量系统，ATP浓度为1mM-5mM，磷酸肌酸和肌激酶[32，33]。肌激酶可选择兔肌激酶，鲤肌激酶。鲤肌激酶M1型(M1-CK)已随体温适应低温环境和PH值。Wu CL等发现，改变兔肌激酶第268位Gly为Asn肌激酶可显著增强肌激酶在低温下催化形成ATP活性。因此理论上优选地，肌激酶选择G268N突变型兔肌激酶或鲤肌激酶[34,35]。
参考NCBI No.AAC96092.1蛋白序列,NP_001075708.1蛋白序列分别合成其基因并分别采用Nde I和EcoR I双酶切克隆至pET22b表达载体，对应的蛋白分别命名为RM-CK和Carp-M1-CK，并在N端融合6xHis标签，以便于纯化。同时参考以上文献，利用常规基因点突变技术，将RM-CK编码基因突变，使其翻译的蛋白氨基酸268位(不含组氨酸标签)G突变为N。
Figure PCTCN2019090586-appb-000008
Figure PCTCN2019090586-appb-000009
扩增的反应条件参考Sinha,N.K.方法，Mg2+离子浓度采用5-20mM，K+离子浓度20-120mM，优选地，浓度为40-80mM；dNTP浓度100uM-1000uM，优选地，300uM-600uM之间。
为进一步提高反应效率，尝试降低温度获得更高反应效率，通过合成DNA定向突变库并筛选，并分别采用Nde I和EcoR I双酶切克隆至pET22b表达载体，表达纯化后获得多株具有体外扩增活性的蛋白突变体，这些突变体蛋白分别命名为VRX_Variant1，VRX_Variant2……依次编号，至VRX_Variant20。其表达的氨基酸序列分别如下：
Figure PCTCN2019090586-appb-000010
Figure PCTCN2019090586-appb-000011
Figure PCTCN2019090586-appb-000012
Figure PCTCN2019090586-appb-000013
Figure PCTCN2019090586-appb-000014
Figure PCTCN2019090586-appb-000015
进一步地，常温扩增反应体系构建如下：
Figure PCTCN2019090586-appb-000016
反应条件如下：25ul，扩增温度：20-45℃。采用水浴锅、恒温设备或PCR仪。反应终点通过琼脂糖凝胶电泳，Sybr green I或特异性探针进行监测。Sybr green I监测时，反应体 系增加终浓度为0.3-0.5x Sybr green I。反应时间可在20-40min，每30s读取一次荧光，荧光通道：FAM/HEX。检测用仪器可使用ABI7500,FTC-3000,Bio-Rad CFX MiniOpticon System,GenDx恒温荧光检测仪GS8等。如采用特异性探针检测扩增体系，则向反应中添加核酸外切酶III或核酸内切酶IV，反应终浓度为50-100ng/ul，荧光标记探针终浓度120nM。探针标记参考[22]进行设计合成。荧光检测采用ABI7500,FTC-3000,Bio-Rad CFX MiniOpticon System,GenDx恒温荧光检测仪GS8等。
本发明的有益效果：将本发明提供的低温噬菌体蛋白应用于常温核酸扩增反应，不仅能够在较低的温度下实现核酸扩增和检测，还能够进一步提高检测的灵敏度，能够检测到100拷贝/ul的核酸。
附图说明
图1为成斑率在不同温度下的试验VR5,VR7,VR20及T4(对照)。
图2为Geneious v5.5基因组进化树分析。
图3为不同物种间uvsX的序列对比。
图4为采用Enterobacteria phage vB_EcoM_VR5扩增体系进行恒温扩增曲线图。
图5为采用Enterobacteria phage vB_EcoM_VR7扩增体系进行恒温扩增曲线图。
图6为采用不同物种来源的混合类型蛋白扩增体系进行恒温扩增曲线图。
图7为利用Enterobacteria phage vB_EcoM_VR5扩增体系和RPA(Recombinase polymerase amplification)技术进行低温扩增所得扩增产物的电泳图，条带1、2、3分别为利用RPA扩增试剂(TALQBAS01)在20℃条件下扩增结果；条带4、5、6分别为利用RPA扩增试剂(TALQBAS01)在25℃条件下扩增结果；条带7、8、9分别为利用Enterobacteria phage vB_EcoM_VR5噬菌体蛋白在20℃条件下扩增结果；条带10、11、12分别为利用Enterobacteria phage vB_EcoM_VR5噬菌体蛋白在25℃条件下扩增结果。
图8为采用突变型肌酸激酶和野生型肌酸激酶扩增体系进行恒温扩增曲线图。
图9为反应体系中采用不同聚合酶进行恒温扩增曲线图。
图10为灵敏度检测曲线图。
图11为反应体系分别采用不同uvsX突变体进行恒温扩增曲线图。
图12为测试不同温度对采用低温噬菌体蛋白的扩增体系扩增效率的影响。
图13为VRX_Variant1 450ng/ul；VR5G_NHis 550ng/ul；VR5Y_NHis 60ng/ul扩增体系对样品检测的恒温扩增曲线图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚，以下结合附图及具体实施方式，对本 发明进行进一步详细说明。以下实施例仅仅用于说明本发明，但不限制本发明的范围。
实施例一构建重组蛋白表达载体及蛋白表达纯化
分别根据NCBI基因组序列(Enterobacteria phage vB_EcoM_VR5,Accession:KP007359.1；vB_EcoM-VR7,Accession:HM563683.1；vB_EcoM_VR20,Accession:KP007360.1；vB_EcoM_VR25,Accession:KP007361.1；vB_EcoM_VR26,Accession:KP007362.1)设计并合成对应的基因序列,并分别采用Nde I和EcoR I双酶切克隆至pET22b表达载体，其中基因序列对应蛋白C末端融合6个组氨酸标签，则命名为基因+CHis，蛋白N末端融合6个组氨酸标签，则命名为基因+NHis，不同病毒株间氨基酸序列一致时则同时加入基因编号，如VR7_25_26Y_CHis，表示vB_EcoM_VR7、vB_EcoM_VR25与vB_EcoM_VR26三株的uvsY氨基酸序列均一致，此编号对应的蛋白为C末端增加了6XHis标签。构建并合成所表达蛋白包括VR5X_NHis，VR7_25X_NHis，VR20_26X_NHis，VR5G_NHis，VR7G_NHis，VR25G_NHis，VR5Y_NHis，VR7_25_26Y_NHis，VR20Y_NHis，VR7_25X_CHis，VR7G_CHis，VR7_25_26Y_CHis等相应的质粒载体，根据分子克隆实验指南技术进行转化宿主细胞BL21(DE3)，IPTG诱导表达，反复冻融裂解并采用Ni柱纯化[27]，获得高纯度蛋白后进行扩增测试。
实施例二Enterobacteria phage vB_EcoM_VR5扩增体系构建
Enterobacteria phage vB_EcoM_VR5扩增体系构建，反应试剂及其浓度如下：三羟甲基氨基甲烷-醋酸缓冲液，30mM；醋酸钾，60mM；醋酸镁20mM；二硫苏糖醇2mM；聚乙二醇(分子量1450-20000)5％；ATP，3mM；磷酸肌酸，30mM；肌酸激酶，90ng/ul；VR5X_NHis蛋白，200-600ng/ul；VR5G_NHis蛋白，200-1000ng/ul；VR5Y_NHis蛋白，60ng/ul；金黄色葡萄球菌聚合酶I大片段(exo-)，8Units；dNTP，450uM；上游引物，250nM；下游引物，250nM；模板为肺炎支原体基因组DNA模板约10ng/ul；Sybr Green I终浓度，0.4x。上游引物peu-F:5'-GCGAACGGGTGAGTAACACGTATCCAATCT-3'(SEQ ID NO.39)250nM；下游引物peu-R1:5'-AGCCATTACCTGCTAAAGTCATTCTTCCCAAA-3'(SEQ ID NO.40)，250nM；反应条件如下：20ul，扩增温度：30℃。采用先达基因恒温荧光扩增仪，型号GS8(http://www.gendx.cn/goods.php？id＝64)。反应终点通过Sybr green I实时检测扩增结果。扩增时间：30分钟。
扩增结果如下
S1:VR5G_NHis蛋白，1000ng/ul VR5X_NHis蛋白，300ng/ul
S2:VR5G_NHis蛋白，800ng/ul VR5X_NHis蛋白，400ng/ul
S3:VR5G_NHis蛋白，600ng/ul VR5X_NHis蛋白，300ng/ul
S4:VR5G_NHis蛋白，400ng/ul VR5X_NHis蛋白，200ng/ul
S5:VR5G_NHis蛋白，200ng/ul VR5X_NHis蛋白，600ng/ul
其它试剂成分与反应条件一致。
扩增结果如图4所示。
结果显示,采用低温蛋白均可以针对特异性模板进行扩增,并通过Sybr Green I嵌合至双链上,发出荧光信号,通过荧光信号读取获得扩增曲线。溶液中不同的低温蛋白浓度扩增效率不一致。
实施例三Enterobacteria phage vB_EcoM_VR7扩增体系构建
Enterobacteria phage vB_EcoM_VR7扩增体系构建，并测试不同末端His标签对蛋白活性的影响。反应试剂及其浓度如下：三羟甲基氨基甲烷-醋酸缓冲液，100mM；醋酸钾，120mM；醋酸镁15mM；二硫苏糖醇6mM；聚乙二醇(分子量1450-20000)6％；ATP，2mM；磷酸肌酸，40mM；肌酸激酶，75ng/ul；VR7_25X_NHis或VR7_25X_CHis蛋白，400ng/ul；VR7G_NHis或VR7G_CHis蛋白，480ng/ul；VR7_25_26Y_NHis或VR7_25_26Y_CHis蛋白，80ng/ul；枯草芽孢杆菌聚合酶I大片段(exo-)，8Units；dNTP，450uM；exo外切酶III，50ng/ul；上游引物ARMP-F，250nM；下游引物ARMP-R，250nM；荧光探针ARMP-PB，120nM；模板为精氨酸支原体基因组DNA模板约25ng/ul；引物与探针序列分别为：ARMP-F:5’-AGCATGTGGTTTAATTTGATGTTACGCGG-3’(SEQ ID NO.41)
ARMP-R:5’-CCATGCACCATCTGTCACTCCGTTAACCTCCG-3’(SEQ ID NO.42)
ARMP-PB:5’-TGTTACGCGGAGAACCTTACCCAC(Fam-dT)(THF)T(BHQ1-dT)GACATCCTTCGCAAT-3’(SEQ ID NO.43)
反应条件如下：50ul，扩增温度：32℃。
采用先达基因恒温荧光扩增仪，型号GS8。扩增结果如下。
S1/S2反应孔：VR7_25X_NHis蛋白，400ng/ul；VR7G_NHis蛋白，480ng/ul；VR7_25_26Y_NHis蛋白，80ng/ul。
S3/S4反应孔：VR7_25X_CHis蛋白400ng/ul；VR7G_CHis蛋白，480ng/ul；VR7_25_26Y_CHis蛋白，80ng/ul
S5反应孔：VR7_25X_NHis蛋白，400ng/ul；VR7G_CHis蛋白，480ng/ul；VR7_25_26Y_NHis蛋白，80ng/ul
S6反应孔：VR7_25X_CHis蛋白，400ng/ul；VR7G_CHis蛋白，480ng/ul；VR7_25_26Y_NHis蛋白，80ng/ul
根据扩增结果显示，尽管扩增的荧光信号高度至有一定差异，扩增荧光信号检测阈值(能够监测到荧光信号值变化的反应时间，TT，Threshold Time)均基本一致。证明在蛋白浓度 相同的情况下，His蛋白标签在融合蛋白的N-末端或C末端对蛋白的活性影响无明显差异。
实施例四不同物种来源的混合类型蛋白扩增体系构建
通过将五种不同病毒株来源的蛋白序列进行混合，测试来源于不同菌株的混合蛋白是否可以进行扩增。反应试剂及其浓度如下：三羟甲基氨基甲烷-醋酸缓冲液，50mM；醋酸钾，80mM；醋酸镁20mM；二硫苏糖醇2mM；聚乙二醇(分子量1450-20000)6％；ATP，2mM；磷酸肌酸，30mM；肌酸激酶，60ng/ul；VR5X_NHis、VR7_25X_NHis或VR20_26X_NHis蛋白，均为400ng/ul；VR7G_NHis或VR25G_NHis蛋白，600ng/ul；VR7_25_26Y_NHis或VR20Y_NHis蛋白，55ng/ul；金黄色葡萄球菌聚合酶I大片段(exo-)，8Units；dNTP，450uM；exo外切酶III，50ng/ul；上游引物susF，400nM；下游引物susR，400nM；荧光探针susPB，120nM；模板为猪总基因组DNA模板约15ng/ul(或者NTC对照，用同等体积量的ddH2O替代)；反应条件如下：50ul，扩增温度：36℃。采用先达基因恒温荧光扩增仪，型号GS8。
引物与探针序列如下：
susF:5’-AGAGATCGGGAGCCTAAATCTCCCCTCAATGG-3’(SEQ ID NO.44)
susR:5’-TCGAGATTGTGCGGTTATTAATGAGTCGTTTGGG-3’(SEQ ID NO.45)
susPB：5’-TGCCACAACTAGATACATCCACATGATTCAT(FAM-dT)(THF)CAA(BHQ1-dT)TACATCAATAAT(C3-SPACER)-3’(SEQ ID NO.46)
结果如图6所示。扩增结果证明，将不同菌株来源的uvsX蛋白、uvsY蛋白、以及GP32蛋白按照一定比例添加至反应中，不同来源蛋白同样可以相互作用，参与引物的结合和解链，并在聚合酶作用下，针对特异性模板进行扩增。尽管蛋白浓度一致，其扩增效率有较大差异，证明不同来源的蛋白其相互作用的能力不一致。
S1/S2：VR5X_NHis VR25G_NHis VR7_25_26Y_NHis
S3/S4：VR5X_NHis VR25G_NHis VR20Y_NHis
S5/S6：VR7_25X_NHis VR25G_NHis VR7_25_26Y_NHis
S7/S8：VR20_26X_NHis VR25G_NHis VR20Y_NHis
其中：S1/S3/S5/S7为添加基因组DNA模板。S2/S4/S6/S8为NTC阴性对照。(图6a)
S1/S2：VR5X_NHis VR7G_NHis VR7_25_26Y_NHis
S3/S4：VR5X_NHis VR7G_NHis VR20Y_NHis
S5/S6：VR5X_NHis VR25G_NHis VR7_25_26Y_NHis
S7/S8：VR20_26X_NHis VR25G_NHis VR7_25_26Y_NHis
其中：S1/S3/S5/S7为NTC阴性对照。S2/S4/S6/S8为添加基因组DNA模板。(图6b)
实施例五利用Enterobacteria phage vB_EcoM_VR5扩增体系测试不同温度对扩增效率的影 响
Enterobacteria phage vB_EcoM_VR5扩增反应试剂及其浓度如下：三羟甲基氨基甲烷-醋酸缓冲液，20mM；醋酸钾，120mM；醋酸镁10mM；二硫苏糖醇8mM；聚乙二醇(分子量20000)5％；ATP，3mM；磷酸肌酸，20mM；肌酸激酶，30ng/ul；VR5X_NHis蛋白，350ng/ul；VR5G_NHis蛋白，500ng/ul；VR5Y_NHis蛋白，50ng/ul；枯草芽孢杆菌聚合酶I大片段(exo-)，10Units；dNTP，450uM；上游引物peu-F:5'-GCGAACGGGTGAGTAACACGTATCCAATCT-3'(SEQ ID NO.47)，250nM；下游引物peu-R1:5'–AGCCATTACCTGCTAAAGTCATTCTTCCCAAA-3'(SEQ ID NO.48)，250nM；模板为携带一段肺炎支原体16srDNA基因序列的质粒模板约100pg/ul；反应条件如下：50ul，扩增温度设置为两个温度：20℃和25℃。RPA技术采用TwistDx试剂(www.twistdx.co.uk产品目录号：TALQBAS01)作为对比，产品使用严格按产品说明书进行。每个试验设置三个重复。采用水浴锅控制反应温度，反应1个小时后，通过立即高温80度蛋白灭活后，扩增产物通过酒精沉淀后进行回收后，利用20ul TE溶解，并取10ul回收产物通过凝胶电泳检测扩增结果。，如图7所示。
携带肺炎支原体16srDNA区段基因序列为:
Figure PCTCN2019090586-appb-000017
序列克隆至pUC57载体上EcoR V酶切平端位点。
通过对比反应图发现，采用本发明的低温物种来源的酶在20℃和25℃低温下扩增效率明显高于利用T4噬菌体uvsX的扩增效率。
根据扩增电泳结果显示,Enterobacteria phage vB_EcoM_VR5噬菌体来源的蛋白扩增效率在低温下明显优于T4噬菌体来源的扩增效率。
实施例六肌酸激酶蛋白突变点对扩增反应的影响
反应试剂及其浓度如下：三羟甲基氨基甲烷-醋酸缓冲液，30mM；醋酸钾，60mM；醋酸镁8mM；二硫苏糖醇4mM；聚乙二醇(分子量1450-20000)3％；ATP，3mM；磷酸肌酸，50mM； RM-CK/RM-CK_G268N/Carp-M1-CK，30-50ng/ul；VR7_25X_NHis蛋白，360ng/ul；VR7G_NHis蛋白，500ng/ul；VR7_25_26Y_NHis蛋白，60ng/ul；枯草芽孢杆菌聚合酶I大片段(exo-)，8Units；dNTP，450uM；上游引物susF，250nM；下游引物susR，250nM；模板为猪肉组织总基因组DNA模板约10ng/ul；采用探针susPB检测，nfo内切酶IV，终浓度为130ng/ul。反应条件如下：25ul，扩增温度：32℃。反应在先达基因GS8荧光扩增仪上进行，荧光扫描间隔60S，反应时间40min。结果如图8所示。
susF:5’-AGAGATCGGGAGCCTAAATCTCCCCTCAATGG-3’(SEQ ID NO.50)
susR:5’-TCGAGATTGTGCGGTTATTAATGAGTCGTTTGGG-3’(SEQ ID NO.51)
susPB：5’-TGCCACAACTAGATACATCCACATGATTCAT(FAM-dT)(THF)CAA(BHQ1-dT)TACATCAATAAT(C3-SPACER)-3’(SEQ ID NO.52)
S1:RM-CK_G268N/50ng/ul
S2:RM-CK_G268N/30ng/ul
S3:RM-CK/50ng/ul
S4:RM-CK/，30ng/ul；
S5:Carp-M1-CK/50ng/ul
S6:Carp-M1-CK，30ng/ul
试验表明，在采用本发明的酶的反应体系中，268位G突变为N的突变体扩增效率优于野生型蛋白RM-CK。
实施例七不同聚合酶对扩增效率的影响
反应体系为：VR7_25X_NHis蛋白，300ng/ul；VR7G_CHis蛋白，400ng/ul；VR7_25_26Y_NHis蛋白,50ng/ul，聚合酶采用金黄色葡萄球菌聚合酶I大片段(exo-)/枯草芽孢杆菌聚合酶I大片段(exo-)/大肠杆菌klenow聚合酶大片段(exo-)/荧光假单胞杆菌聚合酶I大片断(exo-)均为100ng/ul，其它反应试剂及其浓度同实施案例五，另外增加Sybr Green I 0.4X，扩增温度：33℃。反应在先达基因GS8荧光扩增仪上进行，荧光扫描间隔30S，反应时间20min。扩增结果如图9所示。
S1/S2：金黄色葡萄球菌聚合酶I大片段(exo-)
S3/S4：枯草芽孢杆菌聚合酶I大片段(exo-)
S5/S6：大肠杆菌klenow聚合酶大片段(exo-)
S7/S8：荧光假单胞杆菌聚合酶I大片断(exo-)
其中：S1/S3/S5/S7为添加基因组DNA模板。S2/S4/S6/S8为NTC阴性对照。
通过反应结果，除大肠杆菌klenow聚合酶大片段(exo-)扩增效率略低外，其它三个 DNA聚合酶扩增效率均较高。
实施例八35℃低温下测试扩增灵敏度检测下限
反应试剂及其浓度如下：三羟甲基氨基甲烷-醋酸缓冲液，50mM；醋酸钾，100mM；醋酸镁16mM；二硫苏糖醇2mM；聚乙二醇(分子量1450-20000)6％；ATP，2.5mM；磷酸肌酸，30mM；肌酸激酶，120ng/ul；VR7_25X_NHis蛋白，450ng/ul；VR7G_NHis蛋白，700ng/ul；VR7_25_26Y_NHis蛋白，70ng/ul；金黄色葡萄球菌聚合酶I大片段(exo-)，8Units；dNTP，450uM；上游引物，250nM；下游引物，250nM；模板为草鱼呼肠孤病毒GCRV VP7 protein gene合成的质粒序列，分别稀释至10000000拷贝/ul,1000000拷贝/ul,100000拷贝/ul,10000拷贝/ul,1000拷贝/ul,100拷贝/ul，10拷贝/ul，阴性对照为NTC；反应时添加1ul模板至反应体系中。采用探针检测，exo外切酶III，终浓度50ng/ul。反应条件如下：50ul，扩增温度：35℃。GCRV-I-F203:5’-CCCACGCCAACGTCAAGACCATTCAAGACTCC-3’(SEQ ID NO.53)GCRV-I-PB:5’-CAAATGAAGCCATTCGCTCATTAGTCGAAG(Fam-dT)G(THF)G(BHQ1-dT)GACAAAGCGCAGACC(C3-SPACER)-3’(SEQ ID NO.54)
GCRV-I-R313:5’-TCCAATTCGTGATAGTCTACAGTACGGCTACC-3’(SEQ ID NO.55)
携带草鱼呼肠孤病毒GCRV VP7 protein gene基因序列为:
Figure PCTCN2019090586-appb-000018
序列克隆至pUC57载体上EcoR V酶切平端位点。
反应在先达基因GS8荧光扩增仪上进行，荧光扫描间隔30S，反应时间20min。
S1：10000000拷贝/ul,
S2:1000000拷贝/ul,
S3:100000拷贝/ul,
S4：10000拷贝/ul,
S5:1000拷贝/ul,
S6:100拷贝/ul，
S7:10拷贝/ul，
S8:阴性对照为NTC；
通过试验结果图10表明，S6样本扩增非常明显，对于S7样本，稍有荧光信号增加。因此检测灵敏度可以不低于100拷贝/ul，与其它分子诊断技术检测灵敏度接近，通过针对引物和探针序列优化，应该期望获得更优的效果，以实现单个拷贝的扩增荧光信号的检测。
实施例九uvsX蛋白突变点对扩增反应的影响
反应试剂及其浓度如下：三羟甲基氨基甲烷-醋酸缓冲液，20mM；醋酸钾，120mM；醋酸镁10mM；聚乙二醇(分子量1450-20000)6％；ATP，4mM；磷酸肌酸，45mM；肌酸激酶，90ng/ul；二十种不同突变体uvsX蛋白，450ng/ul；VR7G_CHis蛋白，550ng/ul；VR7_25_26Y_NHis蛋白，60ng/ul；金黄色葡萄球菌聚合酶I大片段(exo-)，120ng/ul；dNTP，450uM；上游引物ARMP-F，400nM；下游引物ARMP-R，400nM；模板为模板为携带一段肺炎支原体16srDNA基因序列的质粒模板约3000copies/ul；Sybr Green I浓度为0.5X；反应条件如下：50ul，扩增温度：34℃。反应在MolARRAY MA-6000荧光定量PCR扩增仪上进行，荧光扫描间隔30S，反应时间20min。peu-F:5'-GCGAACGGGTGAGTAACACGTATCCAATCT-3'(SEQ ID NO.57)peu-R2:5'-CAAAGTTCTTATGCGGTATTAGCTAGTCTT-3'(SEQ ID NO.58)结果如图11(a)-(e)所示。在图11(a)中：
S1:VRX_Variant1 450ng/ul；VR5G_NHis 550ng/ul；VR5Y_NHis 60ng/ul；
S2:VRX_Variant2 450ng/ul；VR5G_NHis 550ng/ul；VR5Y_NHis 60ng/ul；
S3:VRX_Variant3 450ng/ul；VR5G_NHis 550ng/ul；VR5Y_NHis 60ng/ul；
S4:VRX_Variant4 450ng/ul；VR5G_NHis 550ng/ul；VR5Y_NHis 60ng/ul；
S5:VR5X_NHis 450ng/ul；VR5G_NHis 550ng/ul；VR5Y_NHis 60ng/ul；
NTC:VR5X_NHis 450ng/ul；VR5G_NHis 550ng/ul；VR5Y_NHis 60ng/ul；无模板。
在图11(b)中：
S6:VRX_Variant5 450ng/ul；VR7G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；
S7:VRX_Variant6 450ng/ul；VR7G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；
S8:VRX_Variant7 450ng/ul；VR7G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；
S9:VRX_Variant8 450ng/ul；VR7G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；
S10:VR7_25X_NHis 450ng/ul；VR7G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；
NTC:VR7_25X_NHis 450ng/ul；VR7G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；无模板。
在图11(c)中：
S11:VRX_Variant9 450ng/ul；VR25G_NHis 550ng/ul；VR20Y_NHis 60ng/ul；
S12:VRX_Variant10 450ng/ul；VR25G_NHis 550ng/ul；VR20Y_NHis 60ng/ul；
S13:VRX_Variant11 450ng/ul；VR25G_NHis 550ng/ul；VR20Y_NHis 60ng/ul；
S14:VRX_Variant12 450ng/ul；VR25G_NHis 550ng/ul；VR20Y_NHis 60ng/ul；
S15:VR20_26X_NHis 450ng/ul；VR25G_NHis 550ng/ul；VR20Y_NHis 60ng/ul；
NTC:VR20_26X_NHis 450ng/ul；VR25G_NHis 550ng/ul；VR20Y_NHis 60ng/ul；无模板。
在图11(d)中：
S16:VRX_Variant13 450ng/ul；VR7G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；
S17:VRX_Variant14 450ng/ul；VR7G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；
S18:VRX_Variant15 450ng/ul；VR7G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；
S19:VRX_Variant16 450ng/ul；VR7G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；
S20:VR7_25X_NHis 450ng/ul；VR7G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；
NTC:VR7_25X_NHis 450ng/ul；VR7G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；无模板。
在图11(e)中：
S21:VRX_Variant17 450ng/ul；VR25G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；
S22:VRX_Variant18 450ng/ul；VR25G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；
S23:VRX_Variant19 450ng/ul；VR25G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；
S24:VRX_Variant20 450ng/ul；VR25G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；
S25:VR20_26X_NHis 450ng/ul；VR25G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；
NTC:VR20_26X_NHis 450ng/ul；VR25G_NHis 550ng/ul；VR7_25_26Y_NHis 60ng/ul；无模板。
试验结果表明：在不同的突变点情况下，部分突变体扩增效率明显高于野生型uvsX蛋白。如VRX_Variant1，VRX_Variant7，VRX_Variant11，VRX_Variant17，VRX_Variant18等等。另外，根据以上实验推断，其它突变体在与不同的gp32，uvsY蛋白组合中，可能也优于野生型蛋白。
实施例十测试不同温度对扩增效率的影响
反应试剂及其浓度如下：三羟甲基氨基甲烷-醋酸缓冲液，30mM；醋酸钾，60mM；醋酸镁 8mM；二硫苏糖醇4mM；聚乙二醇(分子量20000)5％；ATP，3mM；磷酸肌酸，50mM；RM-CK，30ng/ul；VR7_25X_NHis蛋白，360ng/ul；VR7G_NHis蛋白，500ng/ul；VR7_25_26Y_NHis蛋白，60ng/ul；枯草芽孢杆菌聚合酶I大片段(exo-)，8Units；dNTP，450uM；上游引物susF，250nM；下游引物susR，250nM；模板为猪肉组织总基因组DNA模板约10ng/ul；采用探针检测，探针susPB终浓度为120nM，核酸外切酶III(exo III)，终浓度为70ng/ul。反应条件如下：50ul；另外采用RPA技术采用TwistDx试剂(www.twistdx.co.uk产品目录号：TALQBAS01)，并增加终浓度为70ng/ul核酸外切酶III(exo III)作为对比，其它扩增条件均一致。扩增温度：20-45℃，每隔五度一个温度梯度。共六组反应(20、25、30、35、40、45℃)，反应在先达基因GS8荧光扩增仪上进行，荧光扫描间隔30S，反应时间60min。
实验结果如图12所示：左纵坐标:荧光信号值变化,右纵坐标,反应后扫描荧光值开始改变时间(TT),横坐标,代表不同的温度(其中45℃，两种反应试剂均无检测到扩增，故未列出).柱状图:不同温度下扩增后荧光信号变化值.其中灰色代表VR7来源的低温蛋白，黑色为核酸外切酶III(exo III)的RPA扩增试剂；折线图为荧光信号改变时间(TT)，其中灰色代表VR7来源的低温蛋白，黑色为核酸外切酶III。
扩增结果证明，不同于RPA扩增试剂，VR7来源的低温蛋白体系在20-30摄氏度条件下，扩增效果更为明显，而RPA扩增试剂在35-40度扩增效率更高，这与文献报道一致，而本次试验中，20摄氏度条件下，RPA试剂未检测到扩增的荧光信号值变化。
实施例十一采用VRX_Variant1,VR5G_NHis,VR5Y_NHis蛋白组合扩增检测细胞样本中是否有支原体污染
反应试剂及其浓度如下：三羟甲基氨基甲烷-醋酸缓冲液，100mM；醋酸钾，120mM；醋酸镁15mM；二硫苏糖醇6mM；聚乙二醇(分子量20000)5％；ATP，2mM；磷酸肌酸，40mM；肌酸激酶，75ng/ul；VRX_Variant1 450ng/ul；VR5G_NHis 550ng/ul；VR5Y_NHis 60ng/ul；枯草芽孢杆菌聚合酶I大片段(exo-)，8Units；dNTP，450uM；Sybr Green I 0.4X。上游引物ARMP-F，250nM；下游引物ARMP-R，250nM；荧光探针ARMP-PB，120nM；引物与探针序列分别为：
ARMP-F:5’-AGCATGTGGTTTAATTTGATGTTACGCGG-3’(SEQ ID NO.59)
ARMP-R:5’-CCATGCACCATCTGTCACTCCGTTAACCTCCG-3’(SEQ ID NO.60)
反应条件如下：50ul，扩增温度：32℃。样本为确认被支原体污染的细胞培养液；通过扩增后检测荧光曲线。
样本处理：取500μl细胞上清液(或上述细胞悬浮液)，14000rpm离心6min，去除上清收集沉淀(注意：可用吸头吸净上清)，再加入50μl无菌水，震荡混匀，95℃水浴3min后 轻微震荡混匀，快速离心后DNA模板将被释放至上清中，反应时取2.5ul加入体系中作为模板。
反应在
Figure PCTCN2019090586-appb-000019
Mini Opticon荧光定量PCR仪上进行，荧光扫描间隔30S，反应时间25min。扩增结果如图13所示。
在图13中：
S1:样本一号
S2:样本二号
S3:样本三号
S4:样本四号
S5:样本五号
经扩增验证，均可获得阳性扩增曲线，并根据不同的ct值，初步推断样本一号中污染较为严重。
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Figure PCTCN2019090586-appb-000020
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Figure PCTCN2019090586-appb-000024
Figure PCTCN2019090586-appb-000025
Figure PCTCN2019090586-appb-000026
Figure PCTCN2019090586-appb-000027
Figure PCTCN2019090586-appb-000028
Figure PCTCN2019090586-appb-000029
Figure PCTCN2019090586-appb-000030
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Figure PCTCN2019090586-appb-000033

Claims (10)

  1. 低温噬菌体蛋白在常温核酸扩增反应中的应用，其特征在于低温噬菌体蛋白为uvsX蛋白、uvsY蛋白和gp32蛋白和/或分别与低温噬菌体uvsX蛋白、低温噬菌体uvsY蛋白、低温噬菌体gp32蛋白具有相同功能的突变体蛋白，优选情况下低温噬菌体uvsX蛋白选自SEQ ID No.21-23、30任一条序列或与上述序列具有98％及以上同源性的序列，uvsX突变体蛋白选自SEQ ID No.1-20任一条序列或与上述序列具有98％及以上同源性的序列；低温噬菌体uvsY蛋白选自SEQ ID No.27-29、32任一条序列或与上述序列具有98％及以上同源性的序列；低温噬菌体gp32蛋白选自SEQ ID No.24-26、31任一条序列或与上述序列具有98％及以上同源性的序列。
  2. 一种包含低温噬菌体蛋白的常温核酸扩增反应体系，包括引物对和待检测模板、重组酶，聚合酶，单链DNA结合蛋白，核酸酶，dNTP、拥挤试剂、重组加载蛋白、能量系统和盐离子；所述重组酶优选为低温噬菌体UvsX蛋白、与低温噬菌体uvsX蛋白具有相同功能的突变体或其组合；所述聚合酶选自大肠杆菌klenow聚合酶大片段(exo-)，金黄色葡萄球菌聚合酶I大片段(exo-)，枯草芽孢杆菌聚合酶I大片段(exo-)，荧光假单胞杆菌聚合酶I大片断(exo-)及其这些酶的突变体或大片段的任一种或一种以上的组合；所述单链DNA结合蛋白选自低温噬菌体gp32蛋白、与低温噬菌体gp32蛋白具有相同功能的突变体或其组合；所述核酸酶选自核酸外切酶III，核酸内切酶IV；所述重组加载蛋白选自低温噬菌体UvsY蛋白、与低温噬菌体UvsY蛋白具有相同功能的的突变体或其组合；所述拥挤试剂选自聚乙二醇，聚乙烯醇，右旋糖酐或聚蔗糖的一种或一种以上的组合，其中所述聚乙二醇选自PEG1450，PEG3000，PEG8000，PEG10000，PEG14000，PEG20000，PEG25000，PEG30000；所述能量系统选自ATP或ATP，磷酸肌酸，肌酸激酶的组合；盐离子选自Tris，镁离子或钾离子的任一种或一种以上的组合。
  3. 根据权利要求2所述的常温核酸扩增反应体系，其特征在于体系中聚合酶为金黄色葡萄球菌聚合酶I大片段(exo-)，枯草芽孢杆菌聚合酶I大片段(exo-)，荧光假单胞杆菌聚合酶I大片断(exo-)或其组合。
  4. 根据权利要求2所述的常温核酸扩增反应体系，其特征在于体系中肌酸激酶优选为268位G突变为N的突变体。
  5. 根据权利要求2所述的常温核酸扩增反应体系，其特征在于体系中与低温噬菌体UvsX蛋白具有相同功能的突变体选自SEQ ID No.1-23、30任一条序列或其组合。
  6. 根据权利要求2所述的常温核酸扩增反应体系，其特征在于体系中低温噬菌体gp32蛋白选自SEQ ID No.24-26、31任一条序列或与上述序列具有98％及以上同源性的序列。
  7. 根据权利要求2所述的常温核酸扩增反应体系，其特征在于体系中低温噬菌体UvsY蛋白选自SEQ ID No.27-29、32任一条序列或与上述序列具有98％及以上同源性的序列。
  8. 根据权利要求2所述的常温核酸扩增反应体系，其特征在于该体系反应温度为20-40℃，优选地为25-37℃，反应时间为20-40分钟。
  9. SEQ ID No.1-20所示的蛋白或与其具有相同功能且具有98％及以上同源性的蛋白。
  10. 编码权利要求9所示蛋白的核酸序列。
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CN111560472B (zh) * 2020-04-29 2021-09-03 哈尔滨工业大学 一种用于检测新冠病毒SARS-CoV-2的探针和引物、试剂盒、检测方法及应用
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