CN112980928B - Stem-loop primer-assisted isothermal nucleic acid amplification method - Google Patents

Abstract

A stem-loop primer-assisted isothermal nucleic acid amplification technique, characterized by the following steps: a. primer design of SPA: firstly, designing two common primers, namely a forward primer FP and a reverse primer BP, aiming at a template; b. after the common primers are designed, adding stem-loop sequences at the 5' ends of FP and BP to form a pair of stem-loop primers, namely a forward stem-loop primer SFP and a reverse stem-loop primer SBP; c. preparing SPA primer mixed liquid: the amplification primer of the SPA is formed by mixing common primers FP, BP and stem-loop primer SFP and SBP; d. SPA buffer formulation 2 XSPAMix: weighing potassium chloride, ammonium sulfate, magnesium sulfate heptahydrate, betaine, tris-HCl, dNTP, SYBR green I diluted by 1.0ml by 100 times and Tween-20, fully dissolving, fixing the volume, and subpackaging at-20 ℃ for each tube for preservation; e. SPA reaction liquid preparation: SPA reaction is a 10-50 mu L system. The method has the advantages of simple operation, rapidness, sensitivity, economy, practicability and the like, can realize rapid amplification and detection of nucleic acid, and is particularly suitable for on-site detection and rapid screening.

Description

Stem-loop primer-assisted isothermal nucleic acid amplification method

Technical Field

The invention belongs to the field of molecular biology, and in particular relates to a stem-loop primer-assisted isothermal nucleic acid amplification method.

Background

The nucleic acid amplification technology is one of the most widely applied inspection technologies in life science research, and plays a very important role in the fields of basic research, clinical disease diagnosis, infectious disease prevention and control, food safety, environmental monitoring and the like. Nucleic acid amplification techniques are mainly of two major categories: one is the polymerase chain reaction (Polymerase chain reaction, PCR) based on temperature cycling, the other is the isothermal amplification technique (Isothermal amplification technology, IAT) based on constant reaction temperature.

PCR is the most widely used nucleic acid amplification technology at present, and the amplification principle is simpler, and the amplification reaction can be realized by only one pair of primers and one DNA polymerase. Therefore, the PCR technology is widely applied to basic research and clinical practice after the advent of the PCR technology. However, with the development of medical environments, doctors and patients have put demands on nucleic acid detection faster, more accurate, more economical, etc. The disadvantages of PCR also become very pronounced. Because PCR techniques require expensive temperature cycling to achieve amplification reactions, and specialized technicians are required to achieve nucleic acid detection. This makes PCR difficult for bedside detection, field detection, home detection and detection in economically lagging areas. Since rapid detection and field detection are important for basic diagnosis and prevention and control of infectious diseases, isothermal amplification technology with the characteristics of simplicity, rapidness, sensitivity, high efficiency, economy and the like has received high attention in recent years.

The isothermal amplification technology is a technology for amplifying and detecting nucleic acid at constant temperature, and has the characteristics of simplicity, rapidness and high efficiency. Over the last two decades, more than ten isothermal amplification techniques have been invented, including loop-mediated isothermal amplification (LAMP), recombinase Polymerase Amplification (RPA), helicase-dependent isothermal amplification (HDA), rolling Circle Amplification (RCA), isothermal amplification based on Nucleic Acid Sequences (NASBA), strand Displacement Amplification (SDA), etc. Unfortunately, these isothermal amplification techniques have several drawbacks and therefore are not widely used in clinical practice.

LAMP is a relatively widely used isothermal amplification technique at present, and typical disadvantages of LAMP are complex primer design, large primer number and poor amplification specificity. The efficient LAMP amplification requires designing 6 primers for eight sections of the template, which not only makes the design of the primers difficult, but also further results in stronger background amplification and poorer specificity of the LAMP technology. The large number of primers not only can easily lead to primer dimers, but also can increase the probability of primer mismatch with background nucleic acid, thereby leading to nonspecific amplification.

RPA is also a widely used isothermal amplification technique in recent years, but RPA amplification requires at least 5 proteins, two primers and one probe (SauDNA polymerase, recA recombinase, single-stranded DNA binding protein, T4 UvsY protein and creatine kinase, pre-primer, post-primer and THF-labeled fluorescent probe) to be achieved. Therefore, the RPA reagent has a complex composition and is expensive. HAD requires modification of dNTP substrates and non-specific amplification is evident. RCA can only amplify circular templates. There are some drawbacks to the existing isothermal amplification techniques. Therefore, the development of a more sensitive, specific, simple, reliable, economical and practical isothermal amplification technology is expected to realize wide clinical application.

The closest prior art to the invention is LAMP technology, and the technical principle and implementation scheme are as follows:

technical principle of LAMP:

loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification technique invented by Notomi et al in 2000. Basic versions of LAMP require at least 4 primers (whose sequences originate from 6 regions of the template) to achieve amplification. As shown in FIG. 1, the 6 template regions involved in the basic LAMP primer were: f1, F2, F3, B1, B2 and B3, the 4 amplification primers consisting of these 6 regions are: front Inner Primer (FIP), rear inner primer (BIP), front outer primer (F3) and rear outer primer (B3). In addition, in order to increase the reaction speed, a pair of accelerating primers, namely a forward accelerating primer (LF) and a reverse accelerating primer (LB), namely an accelerating LAMP, may be added.

The amplification principle of basic LAMP is shown in figure 2: the double-stranded DNA is in a dynamic equilibrium state of melting-renaturation at about 65 ℃, and the inner primer (comprising FIP and BIP), the outer primer (comprising F3 and B3) and the template are annealed, hybridized and extended under the catalysis of Bst DNA polymerase with strand displacement activity (which is the functional basis of isothermal amplification) to form a dumbbell-shaped intermediate product. The dumbbell-shaped intermediate product can be self-extended and can be used as a template to be amplified by a primer, so that a large amount of stem-loop-like and cauliflower-like amplified products are generated, and exponential amplification is initiated. In the accelerated version of LAMP, the accelerating primer is combined to a loop single strand of the stem-loop structure of an amplified product, and then extension and strand displacement reactions are carried out under the catalysis of Bst DNA polymerase, so that more stem-loop-like products are amplified, and the reaction speed is increased. Specific LAMP amplification and typical amplification products thereof are shown in FIG. 3: the positive control was "S-shaped" in amplification curve and produced a large amount of ladder-like specific amplification product, whereas the positive control was devoid of amplification curve and amplification product. However, when the primer design is not good enough, non-specific amplification of the negative control of LAMP easily occurs, as shown in FIG. 4.

LAMP, however, has the following disadvantages: (1) The primer design of LAMP is complex, and when the DNA to be detected is short, it is difficult to design an appropriate LAMP primer. (2) The number of primers required for LAMP amplification is large, and non-specific amplification is easily caused by non-preferred primers.

Disclosure of Invention

The technical scheme provides a novel isothermal amplification method, namely Stem-loop primer assisted isothermal nucleic acid amplification (Stem-loop-primer assisted isothermal amplification, SPA). SPA has the advantages of simple operation, rapidness, sensitivity, economy, practicability and the like, can realize rapid amplification and detection of nucleic acid, and is particularly suitable for on-site detection and rapid screening. The specific technical scheme is as follows:

a stem-loop primer assisted isothermal nucleic acid amplification technology comprises the following steps:

a. primer design of SPA:

two common primers, forward Primer (FP) and reverse primer (BP), were first designed for the template. Primer design was performed with reference to the PCR primer design rules.

In the step a, in order to reduce the probability of primer dimer formation between FP and BP, the ΔG of the 3' -end primer dimer should be less than 2.0 kcal/mol, and the Tm values of FP and BP should be 45-55deg.C, and the amplified product lengths of FP and BP should be 40BP to 80BP.

b. After the common primers are designed, stem-loop sequences are added to the 5' ends of FP and BP to form a pair of Stem-loop primers, namely a forward-loop primer (SFP) and a reverse-loop primer (SBP).

In the step b, the stem length of the stem-loop sequence is generally 6BP to 14 BP, so that the formation of a nucleic acid secondary structure between the stem-loop sequence and FP and BP is avoided, and the nucleic acid secondary structure can be predicted by analysis through online software Mfold (http:// unafild. Rna. Albany. Edu).

c. Preparing SPA primer mixed liquid: the amplification primers for SPA were mixed with common primers (FP and BP) and stem-loop primers (SFP and SBP).

In the step c, the proportion of the stem-loop primer in the primer mixture is varied from 1% to 50%. The concentration of the mixed primer was 3-6. Mu.M.

d. SPA buffer formulation (2 XSPAMIX):

0.149 g potassium chloride, 0.264 g ammonium sulfate, 0.395 g magnesium sulfate heptahydrate, 15g betaine, 3.0 mL Tris-HCl (1.5M, pH=8.5), 10.0 mL 25 mM dNTP,1.0mL SYBR green I diluted 100 times, 2.0 mL Tween-20 were weighed, fully dissolved and sized to 100 mL, and 2.0 mL split-packed per tube and stored at-20 ℃.

e. SPA reaction liquid preparation:

the SPA reaction is a 10-50 system, preferably a 25 [ mu ] L system: including 2 x SPAMIX of 12.5 mu L, 0.5-1 mu L Bst DNA polymerase (8U/mu L), primer mixture of 2-5 mu L, template that awaits measuring, moisturizing to 25 mu L at last. The reaction tube was incubated (55-65 ℃) for 60-120 minutes with a real-time fluorescent thermostat.

The amounts of the primer, the template, the enzyme and other components in the reaction, the reaction time and the reaction temperature are all required to be adjusted according to specific targets to be detected and experimental conditions.

The amplification principle of the invention is shown in FIG. 5:

the invention firstly simplifies the primer design: in the LAMP technology in the prior art, 4 primers are designed at least for 6 areas of a template, so that isothermal amplification can be realized. The invention adopts a brand-new primer design principle, which is called a novel isothermal nucleic acid amplification technology (Stem-loop-primer assisted isothermal amplification, SPA) assisted by Stem-loop primers for short. The primers for SPA are very simple to design, and only 1 pair of common primers, namely a Forward Primer (FP) and a reverse primer (BP) are designed for 2 regions of the template. Then 1 pair of Stem-loop primers, namely a forward-loop primer (SFP) and a reverse-loop primer (SBP), are constructed on the basis of the common primers. The stem-loop primers SFP and SBP are derived from FP and BP, and only a section of general stem-loop sequence is added at the 5' end of the stem-loop primers SFP and SBP. Thus, the 3' -end sequences of SFP and SBP are completely identical to FP and BP, and the binding region of the template is also completely identical. The amplification primer of SPA is formed by mixing the common primer and the stem-loop primer according to a certain proportion.

The invention reduces the occurrence probability of nonspecific amplification: isothermal amplification techniques are relatively prone to non-specific amplification, which is mainly caused by primer dimers. As with the LAMP technique mentioned previously, 4 or 6 primers are required to achieve amplification (consisting of only 6 or 8 DNA fragments, respectively), thus making primer dimer formation extremely easy and leading to nonspecific amplification. However, SPA can be amplified isothermally by only 4 primers consisting of only 2 template fragments, which greatly reduces the chance of primer dimer formation and thus reduces SPA non-specific amplification.

The technical scheme of the invention has the beneficial effects that:

the isothermal amplification technology is a nucleic acid amplification technology reacting at a constant temperature, and has the characteristics of rapidness, sensitivity, economy, no need of a thermal cycler and the like, so that the defects of PCR are greatly overcome. Isothermal amplification technology is an ideal technology for realizing rapid detection and field detection of nucleic acid, and is also an important development direction in the field of molecular diagnosis. LAMP is a relatively widely used isothermal amplification technology at present, and has the remarkable advantages of high sensitivity, high reaction speed and good economy. LAMP, however, also has significant drawbacks: the primer design is complex, the primer number is large, and the amplification specificity is poor. The efficient LAMP amplification requires designing 6 primers for eight sections of the template, which not only makes the design of the primers difficult, but also further results in stronger background amplification and poorer specificity of the LAMP technology. The reason for this is that many primers are not only extremely prone to form primer dimers, but also increase the probability of primer mismatch with background nucleic acid, and many primers are not only extremely prone to cause primer dimers, but also increase the probability of primer mismatch with background nucleic acid, thus causing non-specific amplification.

Compared with the LAMP isothermal technology, the SPA of the technical scheme is a isothermal amplification technology which is simpler and easier to implement, sensitive, specific and economical. The primers of the SPA are simple in design, and rapid and sensitive isothermal amplification of nucleic acid can be realized by only designing a pair of common primers and stem-loop primers, so that rapid detection of pathogenic microorganisms is realized. In view of its simple, rapid, sensitive, economical nature, SPA would be expected to be an ideal portable molecular diagnostic technique. Therefore, the SPA technology has wide application prospect.

In addition, the market scale in the field of molecular diagnostics is enormous, and POCT is the future development direction of in vitro diagnostics. The visualized SPA isothermal amplification technology is a simple, convenient and economic molecular diagnosis technology, and is expected to play an important role in the fields of bedside detection, field detection and home detection. The visualized SPA isothermal amplification technology not only can be used for detecting the novel coronaviruses, but also has wide application value in the whole molecular diagnosis field. Therefore, the technology has important clinical significance, wide application prospect and great social and economic benefits.

Drawings

FIG. 1 is a schematic diagram of a primer design for a LAMP of the prior art;

FIG. 2 is a schematic diagram of amplification principle of the LAMP of the prior art of the present invention;

FIG. 3 is a gel electrophoresis analysis chart of specific amplification and amplification products of LAMP of the prior art;

FIG. 4 shows the non-specific amplification of the LAMP of the prior art according to the invention;

FIG. 5 is a schematic illustration of the amplification principle of SPA of the present invention;

FIG. 6 shows SPA-amplified ALDH2 gene fragment in example 1 of the present invention;

FIG. 7 is a schematic diagram showing the enzymatic cleavage of the ALDH2 amplification product of example 1 of the present invention;

FIG. 8 shows the digestion and electrophoresis analysis of example 1 of the present invention;

FIG. 9 is a sequencing analysis of SPA amplification products of example 1 of the present invention;

FIG. 10 is a schematic illustration of the self-extending function verification of the MDI of example 1 of the present invention;

FIG. 11 is a schematic diagram of SPA primer and LAMP primer in example 1 of the present invention;

FIG. 12 is a comparison of the amplification effects of SPA and LAMP in example 1 of the present invention;

FIG. 13 is a comparison of SPA and LAMP amplification products in example 1 of the present invention;

FIG. 14 is the sensitivity of SPA in example 1 of the present invention;

FIG. 15 is the SPA specificity of example 1 of the present invention;

FIG. 16 is a SPA assay for HPV16 DNA in example 2 of the present invention.

Description of the embodiments

Example 1:

a stem-loop primer assisted isothermal nucleic acid amplification technology is specifically implemented according to the following steps:

acetaldehyde dehydrogenase 2 (Acetaldehyde dehydrogenase, ALDH 2) is a key enzyme in the metabolic process of alcohol and nitroglycerin, and enzymatic activity is lost after ALDH2 mutation, thereby causing alcohol and nitroglycerin metabolic disorder. Therefore, detection of the ALDH2 gene is of great clinical significance. In this example, the nucleic acid sequence is given as follows for a chemically synthesized ALDH2 gene fragment (shown as sequence 13 in the sequence listing) as the template: 5'-CCGGGAGTTGGGCGAGTACGGGCTGCAGGCATACACTGAAGTGAAAACTGTGAGTGTGG-3' (from the reference sequence GenBank: AH 002599.2).

a. Primer design of SPA:

first, two normal forward and reverse primers (FP and BP) are designed for a target nucleic acid (sequences 2 and 3 in the sequence listing): common primer FP (5'-CCGGGAGTTGGGCGAG-3') and common primer BP (5'-CCACACTCACAGTTTTCAC-3').

In the step a, tm values of FP and BP were 53℃and 49℃respectively, and the amplified product lengths of FP and BP were 59 BP.

b. After the common primers were designed, stem-loop sequences were added to the 5' ends of FP and BP to form stem-loop primers (SFP and SBP): hairpin primer SFP (5'-tttatatatatataaaCCGGGAGTTGGGCGAG-3') and hairpin primer SBP (5'-cctatatatatataggCCACACTCACAGTTTTCAC-3') (shown as sequences 4 and 5 in the sequence Listing).

In the step b, the stem length of the stem-loop sequence is 8 BP, so that the stem-loop sequence and FP and BP are prevented from forming a nucleic acid secondary structure.

c. Preparing SPA primer mixed liquid: the amplification primers for SPA were mixed with common primers (FP and BP) and stem-loop primers (SFP and SBP).

In the step c, the proportion of the stem-loop primer in the primer mixture is 40%. The concentration of the mixed primer was 5. Mu.M.

d. SPA buffer formulation (2 XSPAMIX):

0.149 g potassium chloride, 0.264 g ammonium sulfate, 0.395 g magnesium sulfate heptahydrate, 15g betaine, 3.0 mL Tris-HCl (1.5M, pH=8.5), 10.0 mL 25 mM dNTP,1.0mL SYBR green I diluted 100 times, 2.0 mL Tween-20 were weighed, fully dissolved and sized to 100 mL, and 2.0 mL split-packed per tube and stored at-20 ℃.

e. SPA reaction liquid preparation:

SPA reaction generally adopts a 25 mu L system: including 2 x SPAMix of 12.5 mu L,1 mu L Bst DNA polymerase, 4 mu L primer mixture, 2 mu L template (1 x 106 copies/mu L), moisturizing to 25 mu L at last. Negative controls were also set.

The following experimental verification was performed in this example:

the feasibility and the principle correctness of the SPA are verified by real-time fluorescence analysis, enzyme digestion-electrophoresis analysis and product sequencing analysis.

(1) Real-time fluorescence analysis of SPA amplification procedure:

the prepared reaction liquid is placed in a real-time fluorescence PCR instrument to be incubated at the constant temperature of 63 ℃ for reaction for 90 minutes, the change of fluorescence intensity in the reaction process is monitored by real-time fluorescence, the real-time fluorescence result of the SPA amplified ALDH2 gene is shown in figure 6, and the SPA can amplify the ALDH2 gene efficiently.

(2) Cleavage-electrophoresis analysis of SPA amplification products:

the ALDH2 gene fragment of this example contains PstI restriction enzyme sites as shown in FIG. 7, and therefore, the amplified product was further subjected to cleavage and electrophoresis analysis using PstI enzyme to observe the specificity of the amplified product and the accuracy of the amplification mechanism. According to the reaction principle, the SPA amplification product of ALDH2 can be specifically digested with PstI enzyme and formed into four short fragments of different lengths, as shown in FIG. 7. The analysis by enzyme digestion-electrophoresis showed that SPA amplified products were ladder-like DNA of varying lengths, as shown in lane 1 of FIG. 8, and were degraded by PstI enzyme into four short fragments of varying lengths, as shown in lane 1 of FIG. 8. Thus, the specificity of the SPA amplification products and the correctness of the amplification mechanism are further demonstrated.

(3) SPA amplification product sequencing analysis:

to further examine whether the amplified product contains stem-loop sequence DNA, the shortest amplified product in lane 1 of FIG. 8 was excised and recovered, followed by sequencing analysis. The sequencing results are shown in FIG. 9, and the shortest product fragment is a specific amplification product with stem-loop sequences at both ends, which we named dumbbell-shaped intermediates (Monomeric dumbbell intermediate, MDI) per unit length. The base sequence of MDI is identical to that of the original template (except for the stem-loop sequence), which further confirms the specificity of the amplified product and the correctness of the amplification principle.

(4) Verifying the self-extension function of the stem-loop sequence DNA (shown as sequences 2 and 3 in the sequence table):

to further verify the self-extending function of the stem-loop sequence DNA, the shortest product fragment (MDI) in the SPA amplification product was excised and recovered, followed by self-extension with BstDNA polymerase. The results show that BstDNA polymerase catalyzes MDI self-extension to form longer amplification products without the addition of primers, as shown in lane 3) of FIG. 10. This further demonstrates the self-elongation ability of the intermediate with stem-loop sequence and the correctness of the SPA amplification mechanism.

The SPA amplification principle was verified by the above experiments:

FIG. 6 is a SPA amplified ALDH2 gene fragment of example 1 of the present invention; curve 1, positive control; curve 2, no template negative control; curve 3, no primer negative control;

FIG. 7 is a schematic diagram showing the enzymatic cleavage of the ALDH2 amplification product of example 1 of the present invention;

FIG. 8 shows the digestion and electrophoresis analysis of example 1 of the present invention; lane 1, amplification product of curve 1 in fig. 6; lane 1. Amplification products of curve 1 in fig. 6 were digested with Pst i; lane 2, amplification product of curve 2 in fig. 6; lane 3, amplification product of curve 3 in fig. 6; lane 4, PCR amplification products with SFP and SBP as primers; lane 4, PCR amplification products digested with psti;

FIG. 9 is a sequencing analysis of SPA amplification products of example 1 of the present invention; the shortest fragment in lane 1 of FIG. 7 was excised and recovered and analyzed by sequencing. The results show that the shortest fragment is a unit length primary product (Monomeric dumbbell intermediate, MDI) with stem loop sequences at both ends;

FIG. 10 is a schematic illustration of the self-extending function verification of MDI according to example 1 of the present invention; lane 3 (MDI and Bst enzyme only) shows a number of large molecular weight amplification products, indicating MDI self-extension.

Non-specific amplification of SPA is lower than LAMP:

the typical characteristic of SPA is that the primer design is simple and easy to implement, and the non-specific amplification is not easy to occur, and the reason is that: compared with the classical LAMP isothermal amplification technology, the SPA is a simplified version, so that the number of primers is reduced, and the self-extension capability of a stem-loop structure is fully utilized. In the LAMP reaction, because of the large number of primers, primer dimers and primer-background mismatches are easily caused, and these nonspecific pairs will be amplified in subsequent amplification reactions, resulting in higher background and nonspecific amplifications. The number of primers for SPA is relatively small, and thus it is expected that the occurrence of the above-described situation can be greatly reduced. To verify the effect and advantage of SPA in reducing non-specific amplification, we designed closely related SPA primers and LAMP primers (FIG. 11) (shown as sequences 6-9 in the sequence Listing) on the ALDH2 gene, i.e., F2/B2 of the LAMP primers were completely identical to FP/BP of the SPA primers. While the remaining LAMP primers were designed with reference to their design rules. Then, SPA and LAMP amplification were performed under the same conditions using the same amplification reaction buffer and enzyme. The amplification results are shown in FIG. 12: no significant non-specific amplification occurred within 90 minutes of the SPA negative control, whereas significant non-specific amplification occurred in the LAMP negative control. Further, the amplified products were subjected to electrophoretic analysis, as shown in FIG. 13, no significant nonspecific amplified products were seen for the SPA negative control, while significant nonspecific amplified products were seen for the LAMP negative control.

Comparison of non-specific amplification cases of SPA and LAMP:

FIG. 11 is a schematic diagram of SPA primer and LAMP primer in example 1 of the present invention;

FIG. 12 is a comparison of SPA and LAMP amplification effects in example 1 of the present invention;

FIG. 13 is a comparison of SPA and LAMP amplification products in example 1 of the present invention.

Sensitivity of SPA:

the ALDH2 gene fragment was diluted in a 10-fold gradient to prepare a concentration of 10-100000 copies/. Mu.L. The samples diluted with these gradients were then used to analyze the detection sensitivity of SPA as shown in fig. 14. The results showed that the SPA stably detected about 1X 102 copies of the template molecule in a 25ul reaction system, i.e., the limit of detection (LOD) reached 6.65aM.

The sensitivity of SPA is shown in figure 14.

Specificity of SPA:

to study the amplification specificity of SPA, we used E.coli DNA, human Papilloma Virus (HPV) DNA, hepatitis B Virus (HBV) DNA, EBV DNA, tubercle Bacillus (TB) DNA, hepatitis C Virus (HCV) RNA, etc. as templates, and amplified with SPA primers that amplified ALDH2 gene fragments. As a result, as shown in FIG. 15, SPA primers for amplifying ALDH2 gene fragments were not able to amplify the above-mentioned non-specific nucleic acids, i.e., SPA had good specificity.

FIG. 15 specificity of SPA. In the isothermal amplification process for 90 minutes, the SPA primer for amplifying the ALDH2 gene fragment has no cross reaction with E.coli DNA, HPV-DNA, HBV-DNA, EBV-DNA, TB-DNA, HCV-RNA and the like.

Example 2:

human papillomavirus Type 16 (Type 16 Human Papillomavirus, HPV 16) is obviously related to occurrence and development of cervical cancer, and HPV16 infection rate of cervical cancer patients is up to 57%, so HPV16 detection is one of important detection items for early screening of cervical cancer. The present example is directed to HPV 16L 1 gene fragment (HPV 16-DNA) chemically synthesized as template (shown as sequence 10 in the sequence listing), the nucleic acid sequence being: 5'-CACTGTCTACTTGCCTCCTGTCCCAGTATCTAAGGTTGTAAGCACGGATG-3' (from the reference sequence GenBank: K02718.1).

A stem-loop primer assisted isothermal nucleic acid amplification technology comprises the following steps:

a. primer design for SPA as shown in fig. 16A:

first, two normal forward and reverse primers (FP and BP) were designed for a target nucleic acid (sequences 11 and 12 in the sequence listing): normal primer FP (5'-CACTGTCTACTTGCCTCCT-3'), normal primer BP (5'-CATCCGTGCTTACAACCTT-3').

In the step a, tm values of FP and BP are 51℃and 50℃respectively, and the amplified product lengths of FP and BP are 50 BP.

b. After the common primers were designed, stem-loop sequences were added to the 5' ends of FP and BP to form stem-loop primers (SFP and SBP) (shown as sequences 13 and 14 in the sequence Listing): hairpin primer SFP (5'-tttatatatatataaaCACTGTCTACTTGCCTCCT-3') and hairpin primer SBP (5'-tttatatatatataaaCATCCGTGCTTACAACCTT-3').

In the step b, the stem length of the stem-loop sequence is 8 BP, so that the stem-loop sequence and FP and BP are prevented from forming a nucleic acid secondary structure.

c. Preparing SPA primer mixed liquid: the amplification primers for SPA were mixed with common primers (FP and BP) and stem-loop primers (SFP and SBP).

In the step c, the proportion of the stem-loop primer in the primer mixture is 30%. The concentration of the mixed primer was 5. Mu.M.

d. SPA buffer formulation (2 XSPAMIX):

0.149 g potassium chloride, 0.264 g ammonium sulfate, 0.395 g magnesium sulfate heptahydrate, 15g betaine, 3.0 mL Tris-HCl (1.5M, pH=8.5), 10.0 mL 25 mM dNTP,1.0mL SYBR green I diluted 100 times, 2.0 mL Tween-20 were weighed, fully dissolved and sized to 100 mL, and 2.0 mL split-packed per tube and stored at-20 ℃.

e. SPA reaction liquid preparation:

SPA reaction generally adopts a 25 mu L system: including 12.5 mu L2X SPAMIX,0.8 mu L Bst DNA polymerase, 4 mu L primer mixture, 2 mu L HPV16-DNA solution (1X 106 copies/mu L), moisturizing to 25 mu L at last. Negative controls were also set.

Referring to FIG. 16, SPA amplified HPV16-DNA. (A) template to be detected and SPA primer. (B) real-time fluorescence monitoring of the amplification process. And (C) gel electrophoresis analysis of the amplified product.

The following experiments were performed in this example:

SPA amplification of HPV16-DNA:

the prepared reaction solution is placed in a real-time fluorescence PCR instrument to be incubated at a constant temperature of 63 ℃ for reaction for 90 minutes. The change of fluorescence intensity in the reaction process is monitored by real-time fluorescence so as to observe the change condition of the amplified product of SPA. Real-time fluorescence results of SPA amplification of HPV16-DNA As shown in FIG. 16B, SPA did efficiently amplify HPV16-DNA, and negative control did not undergo non-specific amplification within 90 minutes.

Electrophoretically analyzing the SPA amplification product:

the amplified products were further subjected to gel electrophoresis analysis, and the results are shown in FIG. 16C: the negative control showed no non-specific amplification product, and the positive control had amplification product that was a typical step-like electrophoresis band with the position of the smallest band (84 bp) consistent with the expectation. This suggests that SPA can specifically amplify HPV16-DNA.

Example 3: a stem-loop primer assisted isothermal nucleic acid amplification technology comprises the following steps:

a. primer design of SPA:

two common primers, forward Primer (FP) and reverse primer (BP), were first designed for the template. Primer design was performed with reference to the PCR primer design rules.

In the step a, in order to reduce the probability of primer dimer formation between FP and BP, the ΔG of the 3' -end primer dimer should be less than 2.0 kcal/mol, and the Tm values of FP and BP should be 50℃and the amplified product length of FP and BP should be 40BP.

b. After the common primers are designed, stem-loop sequences are added to the 5' ends of FP and BP to form a pair of Stem-loop primers, namely a forward-loop primer (SFP) and a reverse-loop primer (SBP).

In the step b, the stem length of the stem-loop sequence is 6BP, and the formation of a nucleic acid secondary structure between the stem-loop sequence and FP and BP should be avoided.

c. Preparing SPA primer mixed liquid: the amplification primers for SPA were mixed with common primers (FP and BP) and stem-loop primers (SFP and SBP).

In the step c, the proportion of the stem-loop primer in the primer mixture is 2%. The concentration of the mixed primer was 3. Mu.M.

d. SPA buffer formulation (2 XSPAMIX):

0.149 g potassium chloride, 0.264 g ammonium sulfate, 0.395 g magnesium sulfate heptahydrate, 15g betaine, 3.0 mL Tris-HCl (1.5M, pH=8.5), 10.0 mL 25 mM dNTP,1.0mL SYBR green I diluted 100 times, 2.0 mL Tween-20 were weighed, fully dissolved and sized to 100 mL, and 2.0 mL split-packed per tube and stored at-20 ℃.

e. SPA reaction liquid preparation:

SPA reaction is preferably 25 [ mu ] L system: including 2 x SPAMIX of 12.5 mu L, 0.5-1 mu L Bst DNA polymerase (8U/mu L), primer mixture of 2-5 mu L, template that awaits measuring, moisturizing to 25 mu L at last. The reaction tube was incubated (55 ℃) for 120 minutes with a real-time fluorescent thermostat.

Example 4: a stem-loop primer assisted isothermal nucleic acid amplification technology comprises the following steps:

a. primer design of SPA:

two common primers, forward Primer (FP) and reverse primer (BP), were first designed for the template. Primer design was performed with reference to the PCR primer design rules.

In the step a, in order to reduce the probability of primer dimer formation between FP and BP, the ΔG of 3' -end primer dimer should be less than 2.0 kcal/mol, and the Tm values of FP and BP should be set at 55℃and the amplified product length of FP and BP should be 80BP.

b. After the common primers are designed, stem-loop sequences are added to the 5' ends of FP and BP to form a pair of Stem-loop primers, namely a forward-loop primer (SFP) and a reverse-loop primer (SBP).

In the step b, the stem of the stem-loop sequence is 14 BP, so that the formation of a nucleic acid secondary structure between the stem-loop sequence and FP and BP is avoided, and the nucleic acid secondary structure can be predicted by analysis through online software Mfold (http:// unafile. Rna. Albany. Edu).

c. Preparing SPA primer mixed liquid: the amplification primers for SPA were mixed with common primers (FP and BP) and stem-loop primers (SFP and SBP).

In the step c, the proportion of the stem-loop primer in the primer mixture is 48%. The concentration of the mixed primer was 6. Mu.M.

d. SPA buffer formulation (2 XSPAMIX):

0.149 g potassium chloride, 0.264 g ammonium sulfate, 0.395 g magnesium sulfate heptahydrate, 15g betaine, 3.0 mL Tris-HCl (1.5M, pH=8.5), 10.0 mL 25 mM dNTP,1.0mL SYBR green I diluted 100 times, 2.0 mL Tween-20 were weighed, fully dissolved and sized to 100 mL, and 2.0 mL split-packed per tube and stored at-20 ℃.

e. SPA reaction liquid preparation:

SPA reaction is preferably 25 [ mu ] L system: including 2 x SPAMIX of 12.5 mu L, 0.5-1 mu L LBst DNA polymerase (8U/mu L), primer mixture of 2-5 mu L, template that awaits measuring, moisturizing to 25 mu L at last. The reaction tube was incubated (65 ℃) for 60 minutes with a real-time fluorescent thermostat.

SEQUENCE LISTING

<110> affiliated Hospital of Chuanbei medical college

<120> a stem-loop primer-assisted isothermal nucleic acid amplification method

<130> 2021

<160> 14

<170> PatentIn version 3.3

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tgaaaactgt gagtgtggga cctgctgggg gctcagg 97

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ccgggagttg ggcgag 16

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ccacactcac agttttcac 19

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gcatacactg accacactca cagttttcac 30

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cactgtctac ttgcctcctg tcccagtatc taaggttgta agcacggatg 50

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Claims (1)

1. A non-diagnostic and therapeutic Stem-loop primer-assisted isothermal nucleic acid amplification (Stem-loop-primer assisted isothermal amplification, SPA) method, comprising the steps of:

a. primer design of SPA: firstly, designing two common primers, namely a forward primer FP and a reverse primer BP, aiming at a template;

b. after the common primers are designed, adding stem-loop sequences at the 5' ends of FP and BP to form a pair of stem-loop primers, namely a forward stem-loop primer SFP and a reverse stem-loop primer SBP;

c. preparing SPA primer mixed liquid: the amplification primer of the SPA is formed by mixing common primers FP, BP and stem-loop primer SFP and SBP;

d. SPA buffer formulation 2 XSPAMix: weighing potassium chloride, ammonium sulfate, magnesium sulfate heptahydrate, betaine, tris-HCl, dNTP, SYBR green I diluted by 1.0ml100 times, and Tween-20, fully dissolving, fixing the volume, and subpackaging at-20 ℃ for each tube for preservation;

e. SPA reaction liquid preparation: SPA reaction is carried out in a system of 10-50 mu L;

in the step a, the delta G of the 3' -end primer dimer is less than 2.0 kcal/mol, the Tm value of FP and BP is between 45 and 55 ℃, and the length of amplified products of FP and BP is between 40 and 80 BP;

in the step b, the stem length of the stem loop sequence is 6-14 bp;

in the step c, the proportion of the stem-loop primer in the primer mixture is 1-50%, and the concentration of the mixed primer is 3-6 mu M;

in the step d, 0.149 g potassium chloride, 0.264 g ammonium sulfate, 0.395 g magnesium sulfate heptahydrate, 15g betaine, 3.0 mL 1.5M dNTP of Tris-HCl,10.0 mL 25 mM with pH=8.5, 1.0mL 100-fold diluted SYBR green I, 2.0 mL Tween-20 are weighed, fully dissolved and fixed to 100 mL, and 2.0 mL are packaged for each tube and stored at the temperature of minus 20 ℃;

in the step e, the SPA reaction is a 25 mu L system, which comprises 2X SPA mix of 12.5 mu L, bst DNA polymerase of 0.5-1 mu L8U/mu L, primer mixture of 2-5 mu L, a template to be detected, and finally water is supplemented to 25 mu L, a reaction tube is placed at the temperature of 55-65 ℃ of a real-time fluorescence thermostat, and the reaction tube is incubated for 60-120 minutes.