CN115125290A - Amplification method of multiple single strands and application thereof - Google Patents
Amplification method of multiple single strands and application thereof Download PDFInfo
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Abstract
The invention discloses a multiplex single-chain amplification method and application thereof.A linear primer, an upstream target primer and a downstream target primer are placed in an amplification reaction system for amplification reaction, and the amplification reaction comprises exponential amplification and linear amplification; the upstream target primer and the downstream target primer are respectively a mixture of multiple target primers; the linear primer is a non-target related sequence and is in a half-half complementary hairpin structure form; the upstream target primer is formed by connecting a target specificity sequence and a linear primer sequence in series, wherein the target specificity sequence is at the 3 'end, and the linear primer sequence is at the 5' end. The multiple single-strand amplification method has the advantages of high sensitivity, good specificity, good universality, high multiple single-strand yield, high single-strand yield and good uniformity of yield of each target single strand, and can be applied to nucleic acid hybridization amplification.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a multiplex single-chain amplification method and application thereof.
Background
With the popularization and application of gene detection in modern clinical medical diagnosis, nucleic acid hybridization is increasingly widely applied in various diagnostic detection methodologies, such as gene chip, reverse dot hybridization, second generation sequencing, nucleic acid capture, nucleic acid aptamer screening, Southern hybridization, Northern hybridization and the like.
In nucleic acid hybridization applications, preparation of single-stranded nucleic acids is involved, and is mainly achieved by methods such as chain polymerase amplification (PCR), double-stranded nucleic acid thermal denaturation treatment, double-stranded nucleic acid alkali denaturation treatment, chemical synthesis and the like.
The chain polymerase amplification (PCR) method, one by linear amplification, is limited by the number of templates and generally yields very low. One is amplification by asymmetric PCR, but when a plurality of target primers are simultaneously amplified, the mutual interference is serious, and single-stranded products of each target cannot be covered. The thermal denaturation treatment of double-stranded nucleic acid is complicated in operation and has high requirements for temperature and time control, and denatured single-stranded nucleic acid is easily denatured into double-stranded nucleic acid and has poor stability. The alkali denaturation treatment of the double-stranded nucleic acid has certain destructiveness to the nucleic acid, needs strict pH value control and subsequent pH value neutralization reaction, and is complex to operate. The chemical synthesis of single-stranded nucleic acid is costly, and especially the synthesis of long-fragment single-stranded nucleic acid is difficult.
Therefore, the development of a multiplex single-stranded amplification method which is simple in operation, high in single-stranded yield, free of interference and low in cost is of great significance.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a multiplex single-chain amplification method and application thereof.
The invention is realized by the following technical scheme:
a multiple single-chain amplification method is characterized in that a linear primer, an upstream target primer and a downstream target primer are placed in an amplification reaction system for amplification reaction, and the amplification reaction comprises exponential amplification and linear amplification;
the upstream target primer and the downstream target primer are respectively a mixture of multiple target primers;
the linear primer is a non-target related sequence and is in a half-half complementary hairpin structure form;
the upstream target primer is formed by connecting a target specificity sequence and a linear primer sequence in series, wherein the target specificity sequence is at the 3 'end, and the linear primer sequence is at the 5' end.
Further, the amplification reaction also comprises denaturation treatment.
Further, the sequence length of the linear primer is 30 bp-60 bp, and T is m The value is 56-80 ℃; the upstream target primer has long target specific sequenceThe degree is 14 bp to 22 bp, T m The value is 42-55 ℃; the downstream target primer sequence length is 14 bp-22 bp, T m The value is 42 ℃ to 55 ℃.
Furthermore, the concentration of the linear primer in the amplification reaction system is 0.3-2 mu M, the concentration of the upstream target primer is 0.02-0.6 mu M, and the concentration of the downstream target primer is 0.1-1.2 mu M.
Further, the amplification reaction system meets the condition that the concentration of the linear primer is more than that of the downstream target primer is more than that of the upstream target primer.
Further, the exponential amplification is the first round of amplification, the annealing temperature is 42-55 ℃, and the amplification cycle number is 15-45 cycles; the linear amplification is the second round of amplification, the annealing temperature is 56-72 ℃, and the amplification cycle number is 1-25 cycles.
Further, the denaturation treatment is a third round of amplification, and the heat treatment is carried out at 98 ℃ for 0-15 min and at 4 ℃ for 0-15 min.
Furthermore, the linear primer is modified at the 5' end of the sequence, and the modifying group is one of biotin, digoxin, quantum dots, FAM, VIC, Cy5 and Cy 3.
In the invention, the amplification method of the multiple single strands is applied to nucleic acid hybridization amplification.
According to the invention, the first round of exponential amplification is carried out, and under the condition of low annealing temperature, a large amount of double-stranded primers are generated by target primer amplification; performing second round of linear amplification, wherein under the condition of high annealing temperature, the modified primers are amplified in a large quantity to generate a large quantity of modified single-stranded primers; the third round of denaturation treatment can increase the quantity of single-chain products, and selection can be made according to the actual detection sensitivity requirement.
Advantageous effects
According to the invention, the linear primer, the upstream target primer and the downstream target primer are placed in an amplification reaction system for amplification reaction, and the multiple single-stranded amplification method has the advantages of high sensitivity, good specificity, good universality, high multiple single-stranded yield, high single-stranded yield and high uniformity of the yield of each target single strand.
Drawings
FIG. 1 is a diagram of a probe immobilization array for each HPV type;
FIG. 2 shows HPV16 of human papillomavirus at a concentration of 10 6 、10 2 IU/mL time detection result chart;
FIG. 3 shows HPV18 of human papillomavirus at a concentration of 10 6 、10 2 IU/mL time detection result chart;
FIG. 4 shows HPV31 of human papillomavirus at a concentration of 10 6 、10 2 IU/mL time detection result chart;
FIG. 5 shows HPV33 of human papillomavirus at a concentration of 10 6 、10 2 IU/mL time detection result chart;
FIG. 6 shows HPV35 of human papillomavirus at a concentration of 10 6 、10 2 IU/mL detection result graph;
FIG. 7 shows HPV39 of human papillomavirus at a concentration of 10 6 、10 2 IU/mL time detection result chart;
FIG. 8 shows HPV45 from human papillomavirus at a concentration of 10 6 、10 2 IU/mL time detection result chart;
FIG. 9 shows human papillomavirus HPV51 at a concentration of 10 6 、10 2 IU/mL detection result graph;
FIG. 10 shows HPV52 from human papillomavirus at a concentration of 10 6 、10 2 IU/mL time detection result chart;
FIG. 11 shows human papillomavirus HPV56 at a concentration of 10 6 、10 2 A detection result chart under IU/mL;
FIG. 12 shows human papillomavirus HPV58 at a concentration of 10 6 、10 2 A detection result chart under IU/mL;
FIG. 13 shows HPV59 from human papillomavirus at a concentration of 10 6 、10 2 A detection result chart under IU/mL;
FIG. 14 shows HPV66 from human papillomavirus at a concentration of 10 6 、10 2 A detection result chart under IU/mL;
FIG. 15 shows human papillomavirus HPV68 at a concentration of 10 6 、10 2 A detection result chart under IU/mL;
FIG. 16 shows HPV26 of human papillomavirus at a concentration of 10 6 、10 2 A detection result chart under IU/mL;
FIG. 17 shows HPV53 human papillomavirus at a concentration of 10 6 、10 2 A detection result chart under IU/mL;
FIG. 18 shows HPV73 human papillomavirus at a concentration of 10 6 、10 2 A detection result chart under IU/mL;
FIG. 19 shows HPV82 from human papillomavirus at a concentration of 10 6 、10 2 A detection result chart under IU/mL;
FIG. 20 shows HPV6 from human papillomavirus at a concentration of 10 6 、10 2 A detection result chart under IU/mL;
FIG. 21 human papillomavirus HPV11 at a concentration of 10 6 、10 2 A detection result chart under IU/mL;
FIG. 22 shows human papillomavirus HPV42 at a concentration of 10 6 、10 2 A detection result chart under IU/mL;
FIG. 23 shows human papillomavirus HPV43 at a concentration of 10 6 、10 2 A detection result chart under IU/mL;
FIG. 24 shows human papillomavirus HPV81 at a concentration of 10 6 、10 2 A detection result chart under IU/mL;
FIG. 25 is a diagram showing the results of detection of cross-interferents CMV, HSV-1, CT, GBS, UU and water.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and the described embodiments are only some embodiments, but not all embodiments, of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Amplification and detection of 23 human papilloma virus multiple single chains
(1) Primers for each HPV type were synthesized by Shanghai Biotechnology engineering, Inc. and the sequences are shown in Table 1 below:
TABLE 1 sequence Listing of Linear primers, upstream target primers and downstream target primers for each HPV type
(2) The linear primers (biotin modified at the 5' end of the sequence) were diluted to 10 μ M, each of the upstream and downstream target primers were diluted to 100 μ M, and then each of the upstream and downstream target primers were mixed together. HPV amplification template, human papillomavirus typing national reference from midhospital, diluting the national reference of each type to 10 according to the operation requirement 6 IU/mL、10 2 IU/mL, and simultaneously, respectively extracting nucleic acid by using virus bacteria CMV, HSV-1, CT, GBS and UU as cross-interference substances.
(3) Preparing a 23-fold single-strand amplification reaction system as shown in the following table 2:
TABLE 223 heavy HPV Single Strand amplification reaction System composition
(4) The amplification procedure of the 23-plex HPV single-strand amplification reaction system is shown in table 3 below:
TABLE 323 amplification procedure for heavy HPV Single Strand amplification reaction System
(5) Probe immobilization
Probes for each HPV type were synthesized by Shanghai Bioengineering, Inc. and the sequences are shown in Table 4 below:
TABLE 4 sequence Listing of probes for various HPV types
A probe immobilization kit originated from Shandong anti-Liriomo Biotechnology development Co., Ltd. (1) According to the requirements of the specification, diluting each probe to 50 μ M concentration, mixing and uniformly mixing with a nucleic acid fixing solution according to the volume ratio of 1:4, spotting on a nitrocellulose membrane by using a spotting instrument, baking for 10min at 50 ℃, immersing in a sealing solution, soaking for 10min at 50 ℃, and taking out the nitrocellulose membrane to obtain the nitrocellulose membrane fixed with each probe, wherein the probe fixing array is shown in figure 1:
(6) hybridization assay
The hybridization kit (biotin-streptavidin-alkaline phosphatase method) was from Shandong anti-Haolimo biotechnological development, Inc., and the detection steps are shown in Table 5 below:
TABLE 5 hybridization detection procedure
(7) The result of the detection is that,
various HPV types (HPV 16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68, HPV26, HPV53, HPV73, HPV82, HPV6, HPV11, HPV42, HPV 43) at different concentrations (10 HPV16, HPV18, HPV 43) 6 IU/mL、10 2 IU/mL), and the detection results of the cross-interferents CMV, HSV-1, CT, GBS, UU and water are shown in FIGS. 2 to 25, and it can be seen that the concentration is 10 at a high concentration 6 Under the IU/mL template condition, all HPV types can be correctly detected, cross reaction does not exist among all HPV types, and cross reaction does not exist with CMV, HSV-1, CT, GBS and UU, so that the specificity is good. At a low concentration of 10 2 IU/mL of template conditions, eachThe HPV types can be obviously detected, and the sensitivity is high. In the invention, the 23-fold single-strand amplification can correctly amplify the single-strand products corresponding to the target, and the single-strand products are enough in quantity, high in sensitivity and good in specificity.
Claims (9)
1. A multiple single-chain amplification method is characterized in that a linear primer, an upstream target primer and a downstream target primer are placed in an amplification reaction system for amplification reaction, and the amplification reaction comprises exponential amplification and linear amplification;
the upstream target primer and the downstream target primer are respectively a mixture of multiple target primers;
the linear primer is a non-target related sequence and is in a half-half complementary hairpin structure form;
the upstream target primer is formed by connecting a target specificity sequence and a linear primer sequence in series, wherein the target specificity sequence is at the 3 'end, and the linear primer sequence is at the 5' end.
2. The method for amplifying multiple single strands according to claim 1, wherein the amplification reaction further comprises a denaturing treatment.
3. The method for amplifying multiple single strands according to claim 1, wherein the linear primer has a sequence length of 30bp to 60bp, T m The value is 56-80 ℃; the length of a target specific sequence in the upstream target primer is 14 bp-22 bp, and T m The value is 42-55 ℃; the downstream target primer sequence length is 14 bp-22 bp, T m The value is 42 ℃ to 55 ℃.
4. The method for amplifying a single stranded nucleic acid according to claim 1, wherein the concentration of the linear primer in the amplification reaction system is 0.3 to 2 μ M, the concentration of the upstream target primer is 0.02 to 0.6 μ M, and the concentration of the downstream target primer is 0.1 to 1.2 μ M.
5. The method for amplifying multiple single strands according to claim 4, wherein the amplification reaction system satisfies the conditions of linear primer concentration > downstream target primer concentration > upstream target primer concentration.
6. The method for amplifying a multiple single strand according to claim 1, wherein the exponential amplification is a first round of amplification, the annealing temperature is 42 to 55 ℃, and the number of amplification cycles is 15 to 45 cycles; the linear amplification is the second round of amplification, the annealing temperature is 56-72 ℃, and the amplification cycle number is 1-25 cycles.
7. The method for amplifying a single stranded nucleic acid according to claim 2, wherein the denaturation treatment is a third round of amplification, and the heat treatment is performed at 98 ℃ for 0 to 15 min and at 4 ℃ for 0 to 15 min.
8. The method for amplifying multiple single strands according to claim 1, wherein the linear primer is modified at the 5' end of the sequence, and the modifying group is one of biotin, digoxin, quantum dots, FAM, VIC, Cy5, and Cy 3.
9. Use of the method for amplifying multiple single strands according to any one of claims 1 to 8 in nucleic acid hybridization amplification.
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Citations (6)
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| US20040121364A1 (en) * | 2000-02-07 | 2004-06-24 | Mark Chee | Multiplex nucleic acid reactions |
| US20090291475A1 (en) * | 2008-04-23 | 2009-11-26 | Kai Qin Lao | Sequence amplification with linear primers |
| WO2010147848A2 (en) * | 2009-06-15 | 2010-12-23 | Rd Biosciences, Inc. | Kits and methods for selective amplification and detection of nucleic acid targets |
| CN103614478A (en) * | 2013-12-03 | 2014-03-05 | 浙江农林大学 | Preparation method of single-strand long probe for multiple detection |
| CN110446787A (en) * | 2017-03-24 | 2019-11-12 | 生物辐射实验室股份有限公司 | General clamp primers |
| CN113755558A (en) * | 2021-08-27 | 2021-12-07 | 万子健生物技术(上海)有限公司 | Nucleic acid detection method based on liquid chip technology |
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Patent Citations (6)
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| US20040121364A1 (en) * | 2000-02-07 | 2004-06-24 | Mark Chee | Multiplex nucleic acid reactions |
| US20090291475A1 (en) * | 2008-04-23 | 2009-11-26 | Kai Qin Lao | Sequence amplification with linear primers |
| WO2010147848A2 (en) * | 2009-06-15 | 2010-12-23 | Rd Biosciences, Inc. | Kits and methods for selective amplification and detection of nucleic acid targets |
| CN103614478A (en) * | 2013-12-03 | 2014-03-05 | 浙江农林大学 | Preparation method of single-strand long probe for multiple detection |
| CN110446787A (en) * | 2017-03-24 | 2019-11-12 | 生物辐射实验室股份有限公司 | General clamp primers |
| CN113755558A (en) * | 2021-08-27 | 2021-12-07 | 万子健生物技术(上海)有限公司 | Nucleic acid detection method based on liquid chip technology |
Non-Patent Citations (1)
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