Multipurpose digital PCR (Polymerase Chain Reaction) hypersensitive premixed solution and application thereof

Abstract

The invention discloses a multi-purpose digital PCR (Polymerase Chain Reaction) hypersensitive premixed solution and application, the multi-purpose digital PCR hypersensitive premixed solution comprises 0.1-0.8 U / [mu] L of Taq rapid hot start enzyme, 20-45 mmol of tris-HCl, 20-85 mmol of KCl, 0.5-16 mmol of MgCl2, 25-125 mmol of NH4Cl2, 0.1-1% of glycerol, 0.2-3 mmol of dntp, 0.1-2 mol of betaine, 0.1-1% of Dmso, 30-150 ng / [mu] L of BSA (Bovine Serum Albumin), 0.2-1% of PEG (Polyethylene Glycol) series, 0.01-0.1% of preservative or 0.1-1% of stabilizer, and the percentage is volume fraction. The detection time of the premixed liquid is short and is about 0.33 h, and the liquid drops are still stable and free of fusion phenomenon after being subjected to PCR thermal circulation; 14 severe pneumonia pathogens can be rapidly detected by one tube.

Description

Technical Field

[0001] The present invention relates to a PCR hypersensitive premix, and specifically to a multi-purpose digital PCR hypersensitive premix. Background Art

[0002] The formulation of traditional PCR reaction premixes needs to be determined according to the type and requirements of PCR reactions. Different reaction types and sample types require different reaction premixes, and they have problems such as low detection sensitivity, low accuracy, and poor high tolerance, making it difficult to achieve absolute quantitative analysis of samples. In recent years, with the increasing maturity of gene detection technologies, premixes developed based on digital PCR technology have developed rapidly and have been widely used in gene mutation detection, copy number variation detection, virus and microorganism detection, genetically modified food detection, and sequencing.

[0003] Disadvantages of existing digital PCR premixes: The formulation of the premix needs to be determined according to the type and requirements of PCR reactions. Different reaction types and sample types require different reaction premixes, and they have low detection sensitivity, low accuracy, long PCR amplification time (2 - 4 h), and poor droplet heat resistance stability. Summary of the Invention

[0004] Object of the Invention: The object of the present invention is to provide a multi-purpose digital PCR hypersensitive premix. This premix solves the problem that the formulation of traditional premixes needs to be determined according to the type and requirements of PCR reactions, and different reaction types and sample types require different reaction premixes. The multi-purpose digital PCR hypersensitive premix of the present invention has high detection sensitivity, high accuracy, and high tolerance, a short PCR amplification time period (about 0.33 h), and good high-temperature heat stability of droplets.

[0005] Technical Solution: The multi-purpose digital PCR hypersensitive premix described in the present invention comprises a buffer solution, Taq rapid hot start enzyme, preservative, stabilizer, including 0.1 - 0.8 U / μL Taq rapid hot start enzyme, 20 - 45 mmol tris-HCl, 20 - 85 mmol KCl, 0.5 - 16 mmol MgCl2, 25 - 125 mmol NH4Cl2, 0.1 - 1% glycerol, 0.2 - 3 mmol dNTP, 0.1 - 2 mol betaine, 0.1 - 1% DMSO, 30 - 150 ng / ul BSA, 0.2 - 1% PEG series, 0.01 - 0.1% preservative or 0.1 - 1% stabilizer, and the above percentages are volume fractions.

[0006] Further, the Taq rapid hot start enzyme includes an antibody-modified Taq rapid hot start enzyme or a chemically modified Taq rapid hot start enzyme.

[0007] Further, the PEG series includes one or a combination of more of: PEG1000, PEG1500, PEG2000, PEG3000, PEG4000, PEG6000 or PEG8000.

[0008] Further, the BSA includes one or a combination of more of: conventional BSA, Fatty Acid-Free BSA, Low Endotoxin BSA, IgG-Free BSA, Protease-Free BSA, NuClease-Free BSA, Acetylated BSA, fluorescently labeled BSA, Biotinylated BSA, radioactively labeled BSA or Cross-Linked BSA.

[0009] Further, the preservative includes one or a combination of more of: Sodium Azide, NaN3, ProClin 300, ProClin 950 or Thimerosal.

[0010] Further, the stabilizer includes one or a combination of more of: polyvinyl alcohol, Tween 20, Tween 80, Ficoll or a high molecular weight copolymer of polyoxyethylene.

[0011] Use of the multi-purpose digital PCR hypersensitive premix in the detection of severe pneumonia pathogens, the severe pneumonia pathogens including 14 bacteria: Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus agalactiae, Staphylococcus epidermidis, Neisseria meningitidis, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, Escherichia coli, Enterobacter Cloacae, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia.

[0012] Further, the primer and probe sequences of the 14 bacteria are shown in the following table. Name Forward primer Reverse primer Probe GCGATTGATGGTGATACGGTT Sequence 4 AAACTCGACTTCAATTTTCTTAGC Sequence 5 GGTGTAGAGAAATATGGTCCTGAAGCAAGTGCA Sequence 6 ACGCAATCTAGCAGATGAAGCA Sequence 7 TCGTGCGTTTTAATTCCAGCT Sequence 8 GCCGAAAACGCTTGATACAGGGA Sequence 9 GCAAAAGAACAGATGGAACAAAGTG Sequence 10 CCCCTTACTTCCTTTTTCTATACC Sequence 11 CTGTTGCTACTTACGGCGATTATGG Sequence 12 GTATCTTAGATGATGAAGTTGTTTGT Sequence 13 AAGAATGGAATACATGCTGTTGC Sequence 14 GAAACAACTTGTTTTGGTGAAACGTCCAT Sequence 15 ACGGCACGTGGTACGGTT Sequence 16 GGCTGATTGGCCATTTTTTTCAG Sequence 17 CCGTTTGTTGGCGATATTTCGGTGGTCG Sequence 18 CGATAATAGGTTTGCCTAGTGAG Sequence 19 GGAAGGCTTGACATATGGAAATTG Sequence 20 GCCTAATGAATTCAACGGTTTCACMGB Sequence 21 ACCCAAGTGGACAGACAGA Sequence 22 TTCCATCTTCCCCGTTTGG Sequence 23 AGGAAAAGTGATCAATCCGGGCGAA Sequence 24 AACTTGGGCACAAACACCT Sequence 25 TCCTTGAATTGTTCCATCTTCA Sequence 26 CAGGAAAACGAATCAGTCCTTCGGA Sequence 27 TATTCCGCGATGCTTGTTTTT Sequence 28 ATTATCTCACCAGCAAACTGGCGG Sequence 29 CCCGCAAATCTTTCC MGB Sequence 30 CGTAGATACGGGTGACCGC Sequence 31 AGTTGATCGGGACTTCACAGTAG Sequence 32 TGAAGGCGAAGCAGGCGAGCACGG Sequence 33 TAACGCTACTGCACGTATCGAAGG Sequence 34 TGTATTCGTTTACAAGAGCTGATTT Sequence 35 CACTGGTCCACGTAAGTTGAACGA Sequence 36 CGGCCTGAAATATGACGC Sequence 37 GAAGTCGAACTGATACTGCG Sequence 38 GGCGACCATGTACTCTGAAACCCGC Sequence 39 TTGCAGCAGCCACTCCAA Sequence 40 CTTCGTCAGCCTTGCGA Sequence 41 CCGAAGCTCGTCTGACCGCTACC Sequence 42 AAGAACAAGAGCGAGGTGC Sequence 43 GACAGCTCGATGAACGAGT Sequence 44 GATCGTCGAACGGGTGCACCTGC Sequence 45 Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The currently commercially available premix has disadvantages such as long amplification time and low sensitivity. The 5X multi-purpose digital PCR hypersensitive premix of the present invention has a short detection time, about 0.33 h (total time in the PCR amplification process table in Example 3), and the droplets remain stable without fusion after PCR thermal cycling. 2. The 5X multi-purpose digital PCR hypersensitive premix of the present invention can quickly detect 14 severe pneumonia pathogens in one tube, providing a basis for rapid and accurate treatment in clinical practice. Description of the Drawings

[0013] Figure 1 Figure after droplet amplification in Example 1; Figure 2Diagram after droplet amplification for Example 2; Figure 3 Diagram after droplet amplification for Comparative Example 1; Figure 4 Gene amplification reading result for Candida albicans; Figure 5 For Staphylococcus aureus, Streptococcus pneumoniae, Streptococcusagalactiae, Staphylococcus epidermidis, Neisseria meningitidis Gene amplification reading results (RED) for 5 kinds of bacteria; Figure 6 For Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, Escherichia coli, Enterobacter Cloacae Gene amplification reading results (BLUE) for 5 kinds of bacteria; Figure 7 For Enterobacter Cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia Gene amplification reading results (GREEN) for 4 kinds of bacteria. Detailed implementation manners

[0014] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be further described below.

[0015] Example 1 The specific components of the multi-purpose digital PCR hypersensitive premix or 5X multi-purpose digital PCR hypersensitive premix in this example are as follows: 0.2 U / μL AKTaq DNA Polymerase V2 antibody-modified fast hot start enzyme, 25 mmol tris-HCl, 50 mmol KCl, 0.8 mmol MgCl2, 25 mmol NH4Cl2, 0.3% glycerol, 0.5 mmol dNTP, 1 mol betaine, 0.3% DMSO, 120 ng / ul BSA, 0.5% PEG6000, 0.02% ProClin 950, 0.8% Tween 80 and a high molecular copolymer of polyoxyethylene are compounded in a volume ratio of 1:1 and compounded with the oil phase described in Patent Application No. 2025101545920 to generate droplets and amplify on the Bio-Rad QB200 digital PCR platform. The diagram after droplet amplification is as Figure 1 : As shown in the figure, there is a fusion phenomenon after droplet amplification.

[0016] The above percentages are all volume fractions.

[0017] The preparation method of the above oil phase is as follows: Add perfluoropolyether carboxylic acid with m value = 20 and molecular weight of 4500, dibenzocyclooctyne-polyethylene glycol-amine with n value = 4 and molecular weight of 1000, and lithium perfluorosilicate as a catalyst into a reaction kettle containing fluorinated liquid HFE7500 in a molar ratio of 1:2.05:0.03 successively. Stir at 50 °C for 11 h, cool down to 16 °C and filter by suction. Add 40 g of absolute alcohol to the filtrate, crystallize at 2 °C to precipitate solids, filter and dry to obtain a novel surfactant.

[0018] Add the novel surfactant to fluorinated oil Flou-Oil135 at a mass fraction of 0.2% to prepare a droplet-forming oil.

[0019] Example 2 The specific components of the multi-purpose digital PCR hypersensitive premix or 5X multi-purpose digital PCR hypersensitive premix in this example are as follows: 0.2 U / μL AKTaq DNA Polymerase V2 antibody-modified fast heat-start enzyme, 25 mmol tris-HCl, 50 mmol KCl, 0.8 mmol MgCl2, 25 mmol NH4Cl2, 0.3% glycerol, 0.5 mmol dNTP, 1 mol betaine, 0.3% DMSO, 120 ng / μL BSA, 0.5% PEG6000, 0.02% ProClin 950, 0.8% Tween 80 and a high molecular weight copolymer of polyoxyethylene are compounded in a volume ratio of 1:2 and compounded with the oil phase (the same as in Example 1) described in Patent Application No.: 025101545920, and droplets are generated and amplified on the Bio-Rad QB200 digital PCR platform. After droplet amplification, the figure is as Figure 2 : As shown in the figure, the sizes of the droplets are uniform after amplification and there is no fusion phenomenon.

[0020] All the above percentages are volume fractions.

[0021] Comparative Example 1 (Comparison between Example 2 and Bio-Rad) The specific components of the multi-purpose digital PCR hypersensitive premix of this embodiment or the 5X multi-purpose digital PCR hypersensitive premix are as follows: 0.2 U / μL AKTaq DNA Polymerase V2 antibody-modified fast hot start enzyme, 25 mmol tris-HCl, 50 mmol KCl, 0.8 mmol MgCl2, 25 mmol NH4Cl2, 0.3% glycerol, 0.5 mmol dNTP, 1 mol betaine, 0.3% DMSO, 120 ng / ul BSA, 0.5% PEG6000, 0.02% ProClin 950, 0.8% Tween 80 and a high molecular copolymer of polyethylene oxide are compounded in a volume ratio of 1:2 and compounded with the oil phase (the same as in Example 1) described in Patent Application No. 2025101545920 to generate droplets and amplify on the Bio-Rad QB200 digital PCR platform. The above percentages are all volume fractions.

[0022] Compare the droplets generated and amplified on the Bio-Rad QB200 digital PCR platform using the Bio-Rad consumable Droplet generation oil, part number: 1863005 and ddpcr super mix for probes (no dUTP), part number: 1863023. The comparison diagram is as Figure 3 : It can be seen from the above comparison diagram that the droplets generated by the multi-purpose digital PCR hypersensitive premix of the present invention are still stable and there is no fusion phenomenon after PCR thermal cycling. The droplets generated by the multi-purpose digital PCR hypersensitive premix are as stable as the droplets generated by the Bio-Rad ddpcr super mix for probes (no dUTP).

[0023] Example 3 Add the gene sequence of a Candida albicans to the multi-purpose digital PCR hypersensitive premix described in Example 2, and use it in the stilla Bnaica ® Fully automatic microdroplet chip digital PCR instrument. The amplification process parameters, sequences, and the reading results after amplification are as shown in the following table and Figure 4 shown:

[0024] The gene sequence of Candida albicans is referenced from Zheng Hao, Li Wenge, Yang Haiying, etc. Establishment of a multiplex real-time PCR detection method for common pathogenic bacteria in sepsis [J]. Disease Surveillance, 2017, 32(09): 752-756.

[0025]

[0026] Application Example 1: Add the genes of 14 pathogens of pneumonia Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus agalactiae, Staphylococcus epidermidis, Neisseria meningitidis, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, Escherichia coli, Enterobacter Cloacae, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia It was added to the multi-purpose digital PCR hypersensitivity premix of Example 2 and amplified and read on a Stilla Bnaica ® fully automatic microdroplet chip digital PCR platform. The amplification conditions were as described in the implementation case, and the results after reading were as follows Figure 5 、 6 The sequences of 7 and 14 kinds of bacteria are shown in the following table: Name Amplification Region Forward Primer Reverse Primer Probe GCGATTGATGGTGATACGGTTAAATTAATGTACAAAGGTCATCCAATGACATTCAGACTATTATTGGTTGATACACCTGAAACAAAGCATCCTAAAAAAGGTGTAGAGAAATATGGTCCTGAAGCAAGTGCATTTACGAAAAAAATGGTAGAAAATGCTAAGAAAATTGAAGTCGAGTTT GCGATTGATGGTGATACGGTT Sequence 4 AAACTCGACTTCAATTTTCTTAGC Sequence 5 GGTGTAGAGAAATATGGTCCTGAAGCAAGTGCA Sequence 6 ACGCAATCTAGCAGATGAAGCAGGTTTGCCGAAAACGCTTGATACAGGGAGTTTAGCTGGAATTAAAACGCACGA ACGCAATCTAGCAGATGAAGCA Sequence 7 TCGTGCGTTTTAATTCCAGCT Sequence 8 GCCGAAAACGCTTGATACAGGGA Sequence 9 GCAAAAGAACAGATGGAACAAAGTGGTTCAAAGTTCTTAGGTATTATTCTTAATAAAGTTAATGAATCTGTTGCTACTTACGGCGATTATGGAAATTACGGAAAAAGGTATAGAAAAAGGAAGTAAGGGG GCAAAAGAACAGATGGAACAAAGTG Sequence 10 CCCCTTACTTCCTTTTTCTATACC Sequence 11 CTGTTGCTACTTACGGCGATTATGG Sequence 12 GTATCTTAGATGATGAAGTTGTTTGTCGTTTCCGTGGTAATAACACTGTTATGGCTAAAGAGAAAATGGATTACATGGACGTTTCACCAAAACAAGTTGTTTCAGCAGCAACAGCATGTATTCCATTCTT GTATCTTAGATGATGAAGTTGTTTGT Sequence 13 AAGAATGGAATACATGCTGTTGC Sequence 14 GAAACAACTTGTTTTGGTGAAACGTCCAT Sequence 15 ACGGCACGTGGTACGGTTTCTGTGCCGTTTGTTGGCGATATTTCGGTGGTCGGTAAAACGCCTGGTCAGGTTCAGGAAATTATTAAAGGCCGCCTGAAAAAAATGGCCAATCAGCC ACGGCACGTGGTACGGTT Sequence 16 GGCTGATTGGCCATTTTTTTCAG Sequence 17 CCGTTTGTTGGCGATATTTCGGTGGTCG Sequence 18 CGATAATAGGTTTGCCTAGTGAGATAATCACCACAAACTTGTTTAATCGAAATGTTTTTTGAATGCCTAATGAATTCAACGGTTTCACCAATTTCCATATGTCAAGCCTTCC CGATAATAGGTTTGCCTAGTGAG Sequence 19 GGAAGGCTTGACATATGGAAATTG Sequence 20 GCCTAATGAATTCAACGGTTTCACMGB Sequence 21 ACCCAAGTGGACAGACAGAGGAAGGCTTTACAGGAAAAGTGATCAATCCGGGCGAAATCAAAGAAGAAGGAGCCATCGTTTTTCCAGTTTTACATGGGCCAAACGGGGAAGATGGAA ACCCAAGTGGACAGACAGA Sequence 22 TTCCATCTTCCCCGTTTGG Sequence 23 AGGAAAAGTGATCAATCCGGGCGAA Sequence 24 AACTTGGGCACAAACACCTGAAGAAACAGGCGAATTTTCAGGAAAACGAATCAGTCCTTCGGAAATTTATGAAGAAGAAGCGATTGTTTTCCCTGTTTTACATGGGCCAAATGGTGAAGATGGAACAATTCAAGGA AACTTGGGCACAAACACCT Sequence 25 TCCTTGAATTGTTCCATCTTCA Sequence 26 CAGGAAAACGAATCAGTCCTTCGGA Sequence 27 ATTATCTCACCAGCAAACTGGCGGGGGATACATTTACGCTTGGGTTAAGTATTTCCTATTATCCTAAAAATACGACCTGTAAGCCTGAAAACACAGTTATAAAAGTAGATGATATCGCCTTGTTCCAGCTCAGAAATCAGGGAAAGATTGCGGCGAACAGTAAGGAAGGAACAATTACGTTGAAATGTGATAATCTTTTCGGCGACAAAAAACAAGCATCGCGGAATA TATTCCGCGATGCTTGTTTTT Sequence 28 ATTATCTCACCAGCAAACTGGCGG Sequence 29 CCCGCAAATCTTTCC MGB Sequence 30 CGTAGATACGGGTGACCGCATCCGTCTGGCAGGTGAAGGCGAAGCAGGCGAGCACGGTGCACCAGCAGGCGATCTGTACGTTCAGGTTCAGGTGAAACAGCACGCTATCTTTGAGCGTGAAGGCAACAACCTCTACTGTGAAGTCCCGATCAACT CGTAGATACGGGTGACCGC Sequence 31 AGTTGATCGGGACTTCACAGTAG Sequence 32 TGAAGGCGAAGCAGGCGAGCACGG Sequence 33 TAACGCTACTGCACGTATCGAAGGTCACACAGATAACACTGGTCCACGTAAGTTGAACGAACGTTTATCTTTAGCTCGTGCTAACTCTGTTAAATCAGCTCTTGTAAACGAATACA TAACGCTACTGCACGTATCGAAGG Sequence 34 TGTATTCGTTTACAAGAGCTGATTT Sequence 35 CACTGGTCCACGTAAGTTGAACGA Sequence 36 CGGCCTGAAATATGACGCCAACAATATCTACCTGGCGACCATGTACTCTGAAACCCGCAAGATGACCCCGATCAGCGGCGGCTTTGCCAACAAAGCGCAGAACTTTGAAGCGGTGGCGCAGTATCAGTTCGACTTC CGGCCTGAAATATGACGC Sequence 37 GAAGTCGAACTGATACTGCG Sequence 38 GGCGACCATGTACTCTGAAACCCGC Sequence 39 TTGCAGCAGCCACTCCAAAGAAACCGAAGCTCGTCTGACCGCTACCGAAGACGCAGCTGCTCGTGCTCAGGCTCGCGCTGACGAAGCCTATCGCAAGGCTGACGAAG TTGCAGCAGCCACTCCAA Sequence 40 CTTCGTCAGCCTTGCGA Sequence 41 CCGAAGCTCGTCTGACCGCTACC Sequence 42 AAGAACAAGAGCGAGGTGCTGGCCGCGATCGTCGAACGGGTGCACCTGCCCTTCATGCAGGAACTGGAACGCACCTCCACCGACCAGCGCGACACGCCGGTGCACGACCTGCGCGCGGTGATGATCCACTCGTTCATCGAGCTGTC AAGAACAAGAGCGAGGTGC Sequence 43 GACAGCTCGATGAACGAGT Sequence 44 GATCGTCGAACGGGTGCACCTGC Sequence 45 The above are only the preferred embodiments of the present invention and do not impose any limitation on the present invention. Any person skilled in the art, without departing from the scope of the technical solution of the present invention, makes any form of equivalent replacement or modification and other changes to the technical solution and technical content disclosed by the present invention, which are all within the content of the technical solution of the present invention and still fall within the protection scope of the present invention.

Claims

1. A multi-purpose digital PCR hypersensitivity premix, characterized in that, It includes 0.1 - 0.8 U / μL Taq fast hot start enzyme, 20 - 45 mmol tris-HCl, 20 - 85 mmol KCl, 0.5 - 16 mmol MgCl2, 25 - 125 mmol NH4Cl2, 0.1 - 1% glycerol, 0.2 - 3 mmol dntp, 0.1 - 2 mol betaine, 0.1 - 1% Dmso, 30 - 150 ng / ul BSA, 0.2 - 1% PEG series, 0.01 - 0.1% preservative or 0.1 - 1% stabilizer. The above percentages are volume fractions.

2. The multi-purpose digital PCR hypersensitive premix according to claim 1, wherein The Taq fast hot start enzyme includes antibody-modified Taq fast hot start enzyme or chemically modified Taq fast hot start enzyme.

3. The multi-purpose digital PCR hypersensitivity premix according to claim 2, wherein The PEG series includes one or a combination of more of the following: PEG1000, PEG1500, PEG2000, PEG3000, PEG4000, PEG6000 or PEG8000.

4. The multi-purpose digital PCR hypersensitivity premix according to claim 3, wherein The BSA includes one or a combination of more of the following: conventional BSA, Fatty Acid-Free BSA, Low Endotoxin BSA, IgG-Free BSA, Protease-Free BSA, NuClease-Free BSA, Acetylated BSA, fluorescently labeled BSA, Biotinylated BSA, radioactively labeled BSA or Cross-Linked BSA.

5. The multi-purpose digital PCR hypersensitivity premix according to claim 1, characterized in that, The preservative includes one or a combination of more of the following: Sodium Azide, NaN3, ProClin 300, ProClin 950 or Thimerosal.

6. The multi-purpose digital PCR hypersensitive premix according to claim 1, wherein The stabilizer includes one or a combination of more of the following: polyvinyl alcohol, Tween 20, Tween 80, Ficoll or a high molecular weight copolymer of polyoxyethylene.

7. Use of the multi-purpose digital PCR hypersensitive premix according to any one of claims 1-6 in the detection of severe pneumonia pathogens, wherein the severe pneumonia pathogens include 14 kinds of bacteria: Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus agalactiae, Staphylococcus epidermidis, Neisseria meningitidis, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, Escherichia coli, Enterobacter Cloacae, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia.

8. The use according to claim 7, characterized in that: The primer and probe sequences of 14 kinds of bacteria are shown in the following table. 。

Images