WO2023109032A1

WO2023109032A1 - Multiple nucleic acid detection system, and preparation method therefor and use thereof

Info

Publication number: WO2023109032A1
Application number: PCT/CN2022/097203
Authority: WO
Inventors: 陈嘉昌; 李楚明; 刘向东; 唐海辉; 王维世; 王辉芳; 张源明; 张乾毅; 胡朝晖; 柳俊
Original assignee: 广州市金圻睿生物科技有限责任公司
Priority date: 2021-12-14
Filing date: 2022-06-06
Publication date: 2023-06-22
Also published as: CN114134219A

Abstract

The present invention relates to a multiple nucleic acid detection system, which comprises an amplification primer set and a detection probe set for a target nucleic acid sequence. The detection system comprises a Target probe modified by LNA and a Beacon probe which achieves a fluorescence quenching effect by means of 1-8 consecutive G bases. Moreover, further provided in the present invention is a multiple nucleic acid detection method combined with the above-mentioned multiple nucleic acid detection system and a touchdown PCR program. The method can further reduce non-specific amplification in a PCR reaction, and improve detection sensitivity. The present invention overcomes the limitations of traditional real-time fluorescent quantitative PCR typing, achieves single-tube multiple typing by means of special signals and melting curve analysis, and detects target nucleic acids in a sample with a simpler reaction system and lower detection costs.

Description

A multiple nucleic acid detection system and its preparation method and application

technical field

The invention relates to the field of biomedicine, in particular to a multiple nucleic acid detection system and its preparation method and application.

Background technique

Real-time fluorescent quantitative PCR method is a commonly used nucleic acid detection method in molecular biology. Compared with ordinary PCR method, it is easy to operate and widely used. During the PCR amplification process of the target gene of the sample to be tested, the fluorescent signal generated by the reaction system is monitored in real time by the instrument, and the PCR process is detected in real time. Ordinary PCR amplification technology requires electrophoresis analysis of the product after the amplification is completed. The analysis process is cumbersome and time-consuming. At the same time, the opening of the PCR product may cause contamination of the laboratory environment.

The methods for realizing fluorescent signals are mainly divided into two types: probe method and dye method. The probe method adds fluorophore-labeled oligonucleotide probes to the reaction system and monitors the fluorescent signals released by the probes in real time to realize the detection of fluorescent signals. Specific detection of target sequence; the dye method is to add double-stranded DNA fluorescent dye to the reaction system, and realize the detection of the target sequence by combining the fluorescent dye into the DNA double-stranded minor groove;

In the real-time fluorescent PCR detection mode, PCR amplification and detection of the target sequence are carried out simultaneously without additional steps. Therefore, the real-time detection mode is simple and straightforward. However, the maximum number of target sequences that can be detected by this mode in a single-tube detection is limited by the number of fluorescence detection channels of the real-time PCR instrument, which generally does not exceed 6. Therefore, based on the methodological advantages of simplicity and directness, it is necessary to improve the real-time fluorescent quantitative PCR method in order to detect more target genes in a single tube detection.

US 2013/0109588 A1 discloses a real-time fluorescent PCR assay method that can be used for melting curve analysis, by designing two probes (PTO probe and CTO probe) to realize the detection of the target sequence. When the method described in this patent application is used to perform multiplex real-time PCR that differentiates each target sequence, one PTO probe and one CTO probe need to be designed separately for each target sequence, ie a double number of probes is used. For example, this patent application describes a dual real-time PCR for simultaneous detection of Neisseria gonorrhoeae and Staphylococcus aureus, in which 2 PTO probes and 2 CTO probes are used. In this case, the patent's method is more complicated and expensive compared to traditional multiplex PCR using a single fluorescent probe for each target sequence.

US 2015/0072887 A1 discloses a real-time PCR assay that can be used for melting curve analysis, which uses three probes to achieve real-time detection of the target sequence. However, when the method described in this patent application is used to implement multiplex real-time PCR that needs to distinguish each target sequence, three probes need to be designed for each target sequence, which leads to more complex reaction system and high detection cost .

Contents of the invention

Based on this, one of the objectives of the present invention is to provide a more convenient and efficient multiplex nucleic acid detection system.

Including the following technical solutions:

A multiplex nucleic acid detection system, comprising an amplification primer set for a target nucleic acid sequence and a detection probe set, the detection probe set including a Target probe and a Beacon probe, and the Target probe extends from the 5' end to the 3' The composition of the end is: 5' end region, Target region, 3' end region; the composition of the Beacon probe from the 5' end to the 3' end is: 5' end region, loop region, 3' end region; The 5' end of the 5' end region sequence of the Target probe is modified with LNA, the 3' end of the 3' end region sequence is modified with C3, and the Target region sequence can be reverse complementary to the target nucleic acid sequence; the Beacon probe's The 5' end of the 5' end region sequence is n consecutive guanines, n is an integer of 1-8, the 3' end of the 3' end region sequence is modified with a fluorescent reporter group, and the 3' end region sequence is compatible with The sequence of the 5' terminal region is reverse complementary.

Another object of the present invention is to provide a multiple nucleic acid detection method.

Including the following technical solutions:

Obtain the nucleic acid of the biological sample to be tested;

Mix the above biological sample nucleic acid, DNA polymerase and the above multiple nucleic acid detection system to prepare a PCR reaction system, and perform PCR reaction and melting curve analysis.

Based on in-depth research on gene detection technology, the inventor of the present invention has developed a more convenient and efficient multiple nucleic acid detection system, which includes a dual-probe system of Target probe and Beacon probe. The inventor found through experiments that, through Structural modification of Target probe and Beacon probe, especially designing the 5' end region and 3 end region of the Target probe as non-homologous sequences, which do not bind to the template, and the 5' end region is modified with LNA, improving For the Tm value of the Target probe, the 3' end region provides a CGCG base chain, and then modified C3 to block non-specific extension; the middle Target region can be reverse complementary to the target nucleic acid sequence, thus effectively providing an anchor point for the binding sequence. At the same time, the sequence of the 5' end region of the Target probe is reversely complementary to the loop region of the Beacon probe, and the Beacon probe is naturally coiled in a free state, because the reporter group (R) and the 5-terminal multiple (1-8 1) The distance between the G bases is short and there is no fluorescence (self-quenching molecular beacon probe). The end region is cut off and is in a free state. When the temperature is lower than the Tm value, it can hybridize with the loop region of the Beacon probe, and under the action of the 5'→3' polymerase activity of DNA polymerase, it can be extended and amplified. After being double-stranded, the Beacon probe fluoresces because the distance between the reporter group and multiple G (1-8) bases is far. Therefore, while effectively reducing the cost of probe synthesis and non-specific amplification in the detection reaction, the detection sensitivity, specificity and stability are improved.

Moreover, the detection system eliminates the difference of different amplicons in the amplification product caused by the difference in amplification efficiency between specific primers through the combined use of CLO primers and universal primers. Thereby, the sample detection is realized more stably as a whole.

At the same time, based on the proposal of the above-mentioned multiple nucleic acid detection system, the inventor combined the design of the PCR program, through the design of the PCR program, combined with the use of the landing PCR program, and also proposed a multiple nucleic acid detection method, which can further reduce the number of non-specific nucleic acids in the PCR reaction. Heterosexual amplification improves detection sensitivity. In order to overcome the limitations of traditional real-time fluorescence quantitative PCR typing, through the analysis of special signals and melting curves, multiple typing in a single tube can be realized, and the real-time fluorescent quantitative PCR instrument can be used for testing in a real-time fluorescent quantitative PCR instrument with a simpler reaction system and lower detection cost. Accurate and qualitative detection can be achieved for each target gene in at most 20 kinds of target nucleic acid sequences to be tested in the sample, and the specificity of detection can be guaranteed by interpreting the specific melting peak in the melting curve.

Description of drawings

Fig. 1 is a schematic diagram of the design of each component in the multiplex nucleic acid detection system of the present invention.

FIG. 2 is a comparison chart of the results of detecting influenza A virus using different primer-probe combinations in Example 2.

Fig. 3 is the agarose gel electrophoresis figure of the influenza A virus PCR product that adopts different PCR programs to amplify in embodiment 2.

Fig. 4 is a comparison chart of the detection results of influenza A virus using different Target probes in Example 2.

5 is a comparison chart of the results of detecting influenza A virus using different Beacon probes in Example 2.

Fig. 6 is the partial result that adopts respiratory pathogen typing detection kit to positive quality control product detection in embodiment 3, and wherein, A is the melting curve figure under FAM channel, C is the amplification curve figure under FAM channel, B is The melting curve graph under the VIC channel, D is the amplification curve graph under the VIC channel.

Fig. 7 is the partial result that adopts respiratory pathogen typing detection kit to positive quality control product detection in embodiment 3, and wherein, A is the melting curve figure under ROX channel, C is the amplification curve figure under ROX channel, B is The melting curve under the CY5 channel, D is the amplification curve under the CY5 channel.

Detailed ways

The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions recommended by the manufacturer. Various commonly used chemical reagents used in the examples are all commercially available products.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the technical field of the present invention. Terms used in the description of the present invention are only for the purpose of describing specific embodiments, and are not used to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In addition, as used in the present invention, the term "or" is an inclusive "or" symbol, and is equivalent to the term "and/or", unless the context clearly dictates otherwise.

In order to facilitate the understanding of the present invention, the following will describe the present invention more fully. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the understanding of the present disclosure will be thorough.

Some embodiments of the present invention provide a multiplex nucleic acid detection system, which is characterized in that it includes an amplification primer set and a detection probe set for a target nucleic acid sequence, and the detection probe set includes a Target probe and a Beacon probe, The composition of the Target probe from the 5' end to the 3' end is: 5' end region, Target region, and 3' end region; the composition of the Beacon probe from the 5' end to the 3' end is: 5' end region, loop region, 3' end region; the 5' end of the Target probe 5' end region sequence is modified with LNA, the 3' end of the 3' end region sequence is modified with C3, and the Target region sequence can be combined with the target nucleic acid The sequence is reverse complementary; the 5' end of the 5' end region sequence of the Beacon probe is n consecutive guanines, n is an integer of 1-8, and the 3' end of the 3' end region sequence is modified with a fluorescent reporter Group, the sequence of the loop region is reverse complementary to the sequence of the 5' end region of the Target probe.

In some of these embodiments, the above-mentioned detection probe set includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, Target probes for at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 target nucleic acid sequences and Beacon probes.

In some of these embodiments, the 3' end of the 3' end region of the Target probe is CGCG, so that the Tm value of the Target probe can be effectively increased and the non-specific amplification in the detection process can be reduced.

In some of these embodiments, both the 5' end region and the 3 end region of the Target probe are non-homologous sequences, which do not bind to the template, and the 5' end region is modified with LNA to increase the Tm value of the Target probe, The 3' end region provides a CGCG base chain, and then modified C3 to block non-specific extension; the middle Target region can be reverse complementary to the target nucleic acid sequence, thereby effectively providing an anchor point for the binding sequence.

In some of these embodiments, the sequence length of the 5' end region of the above-mentioned Beacon probe is 5-8 bp, the sequence length of the 3' end region is 5-8 bp, and/or the sequence length of the loop region is 30-50 bp, And it is reverse complementary to the sequence of the 5' end region of the Target probe.

In some of these embodiments, the number n of continuous guanines at the 5' end of the 5' end region sequence of the above-mentioned Beacon probe is an integer of 3-5. It is further preferred that n is 4, so as to effectively provide the fluorescence quenching function.

In some of these embodiments, the above-mentioned set of amplification primers includes a pair of CLO primers, and the composition of each CLO primer in the pair of CLO primers from the 5' end to the 3' end is as follows: 5' end region, loop region, 3' terminal region; the sequence length of the 5' terminal region is 18-25 bp, the sequence length of the 3' terminal region is 10-15 bp, and the sequence length of the loop region is 15-25 bp.

In some of these embodiments, the above-mentioned set of amplification primers includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, CLO primer pairs of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 target nucleic acid sequences .

In some of these embodiments, the above amplification primer set also includes a pair of universal primers, the upstream primer in the pair of universal primers is consistent with the loop region of the upstream CLO primer in the pair of CLO primers; the downstream primer in the pair of universal primers is consistent with The loop regions of the middle and downstream CLO primers of the CLO primer pair are consistent.

In some of these embodiments, the above-mentioned universal primers can amplify all PCR products generated in the first two cycles after the third cycle of PCR amplification, so as to ensure the uniformity of amplification of each target.

In some of these embodiments, in the above multiple nucleic acid detection system, the 3' terminal modified fluorescent reporter group of the 3' end region sequence of the Beacon probe is selected from FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas, Red, LC RED640, Cy5, LC RED705, Alexa Fluor 488, and Alexa Fluor 750.

Some embodiments of the present invention also provide the application of the above multiple nucleic acid detection system in the preparation of reagents or kits for disease diagnosis.

In some of these embodiments, the above-mentioned diseases are infectious diseases caused by cross-infection caused by various pathogens. Preferably, the aforementioned infectious diseases are respiratory tract infection, digestive tract infection, blood infection and/or urinary tract infection and the like. More preferably, it is used for the detection and determination of pathogens in respiratory tract infections with similar symptoms such as cough, which can be influenza viruses such as influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus , Mycoplasma pneumoniae, parainfluenza virus and other infections.

Some embodiments of the present invention also provide a multiple nucleic acid detection method, which includes the following steps:

Obtain the nucleic acid of the biological sample to be tested;

The nucleic acid of the biological sample, the DNA polymerase and the multiple nucleic acid detection system described in any one of claims 1-7 are mixed to form a PCR reaction system, and the PCR reaction and melting curve analysis are carried out.

In some of the embodiments, the type of the target nucleic acid sequence existing in the reaction system is judged by performing melting curve analysis on the product obtained from the above PCR reaction, and multiple nucleic acid detection is realized.

In some of these embodiments, the reaction procedure of the above PCR reaction is touch down PCR. More preferably, when the detection is performed using the touchdown PCR program, in the first 6 cycles, the annealing temperature is decreased by 1° C. for every additional cycle.

In some of these embodiments, the above-mentioned biological samples may be selected from including but not limited to: serum samples, plasma samples, whole blood samples, sputum samples, swab samples, lavage fluid samples, fresh tissue samples, formalin-fixed Paraffin-embedded tissue (FFPE) samples, urine samples, bacterial cultures, viral cultures, cell line cultures, synthetic plasmid samples.

In some of the embodiments, the biological sample nucleic acid is deoxyribonucleic acid or ribonucleic acid. When the biological sample is ribonucleic acid, the reaction system further includes reverse transcriptase, and the reaction procedure further includes reverse transcription PCR.

In some of these embodiments, in the above-mentioned PCR reaction system, the concentration of each CLO primer for different target nucleic acid sequences is the same, and the concentration of each universal primer is also the same, and the final concentration of each universal primer is 5-5% of the final concentration of a single CLO primer. 15 times, more preferably 9 to 11 times, more preferably 10 times.

In some of these embodiments, in the above-mentioned PCR reaction system, the concentration of each Target probe for different target nucleic acid sequences is the same, and the concentration of each Beacon probe is the same, and the final concentration of each Target probe is 50% of the final concentration of the Beacon probe. 1 to 5 times, more preferably 1 to 3 times, even more preferably 2 times.

In some of the embodiments, the above method can detect 1-20 types of target nucleic acid sequences.

In some embodiments, the above method is not limited by theory, and the resolution or precision of melting curve analysis can reach 0.5°C or higher. In other words, melting curve analysis is able to distinguish two melting peaks whose melting points differ by only 0.5°C or less (eg, 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C).

In some of these embodiments, the melting point difference between the amplification products of any two target nucleic acid sequences to be detected that can be detected by the above method may be at least 0.5°C, so that any two target nucleic acid sequences to be detected can be detected by melting Curve analysis to distinguish and distinguish. However, for the purpose of easy differentiation and discrimination, a larger melting point difference between any two target nucleic acid sequences to be detected is preferred in some cases.

In some of these embodiments, the melting point difference between any two target nucleic acid sequences to be detected by the above method can be any desired value (for example, at least 0.5°C, at least 1°C, at least 2°C, at least 3°C, at least 4°C, at least 5°C, at least 8°C, at least 10°C, at least 15°C, or at least 20°C), as long as the difference in melting points can be distinguished and discerned by melting curve analysis.

Some embodiments of the present invention also provide an influenza A virus detection kit, which includes the above multiple nucleic acid detection system for the conserved segment sequence of the influenza A virus M1 gene.

In some of these embodiments, the above-mentioned multiplex nucleic acid detection system includes an amplification primer set and a detection probe set. In the amplification primer set, the sequence of the CLO primer pair is as shown in SEQ ID NO.1 to SEQ ID NO.2 or As shown in SEQ ID NO.7～SEQ ID NO.8; Universal primer pair as shown in SEQ ID NO.5～SEQ ID NO.6 or as shown in SEQ ID NO.9～SEQ ID NO.10; described In the detection probe set, the Target probe sequence is shown in SEQ ID NO.3; the Beacon probe sequence is shown in SEQ ID NO.4 or as shown in SEQ ID NO.11. Further, the CLO primer pair sequence is shown in SEQ ID NO.7～SEQ ID NO.8; the universal primer pair is shown in SEQ ID NO.9～SEQ ID NO.10; in the detection probe set, Target probe The needle sequence is shown in SEQ ID NO.3; the Beacon probe sequence is shown in SEQ ID NO.4.

The present invention will be described in further detail below in conjunction with specific examples.

The composition of embodiment 1 multiplex nucleic acid detection system

1. Primer probe

For the object to be detected, consult relevant professional literature, determine the conserved segment of the nucleic acid sequence of the object to be detected according to literature research, select at least one specific target gene sequence (target nucleic acid sequence), and based on the selected specific target gene sequence, Design upstream oligonucleotide primer CLO-F (Convex loop oligo-Forward, CLO-F), downstream oligonucleotide primer CLO-R (Convex loop oligo-Reverse, CLO-R), Target probe (T probe ) sequence target gene region, artificial sequence Beacon probe (B probe) and universal primer, wherein, CLO-F comprises a sequence complementary to the specific target gene sequence, and CLO-R comprises the same sequence as the specific target gene sequence, The Target probe is divided into three parts according to the turning points at both ends of the complementary target sequence, the 5-terminal region, the target gene region and the 3-terminal region, the target gene region of the Target probe is complementary to the specific target sequence, the 5-terminal region and the 3-terminal region To artificially introduce sequences, the sequences of the 5-terminal regions of all Target probes are different from each other. The details are as follows and shown in Figure 1.

CLO primer: consists of 3 parts. The 5'-end anchor segment consists of 18-25 bases and has a high Tm value; the 3'-end is a specific binding region, which consists of 10-15 bases and has a low Tm value; the middle loop The region is composed of an artificial sequence of 15-25 bases.

Target probe: consists of 3 parts. Both the 5' end and the 3' end are non-homologous sequences, which do not bind to the template, and the 5' modifies LNA to increase the Tm value of the Target probe, and the 3' provides CGCG base chains, and then modifies C3 to seal; the middle target gene region It is the reverse complementary sequence to the template, providing the binding sequence anchor point.

Beacon probe: provide a fluorescent signal release system to realize melting curve analysis. Consists of 3 parts. The 5' end is composed of 5 to 8 bases, and the end is 4 consecutive G bases, which provide the fluorescence quenching function; the loop region is composed of 30 to 50 bases, and is reversed with the artificial sequence at the 5' end of the Target probe. Complementary to the direction; the 3' end consists of 5 to 8 bases, and the end is modified with a fluorescent reporter group. For different target nucleic acid sequences, the end-modified fluorescent reporter groups of the designed Beacon probes are different.

Universal primer: its sequence and structure are consistent with the Loop region of the CLO primer, which can amplify all PCR products generated in the first two cycles after the third cycle of landing PCR amplification to ensure the uniformity of amplification of each target .

2. System preparation and composition (the nucleic acid of the biological sample is ribonucleic acid as an example in the following, if the biological sample is deoxyribonucleic acid, you can refer to the following method to remove the reverse transcription-related components and reaction procedures)

(1) Enzyme: the concentration of reverse transcriptase is 5U/μL-15U/μL, the reverse transcriptase can be murine leukemia reverse transcriptase (MMLV) or Tth enzyme; the DNA polymerase is 5U/μL-15U/μL, DNA polymerase The enzyme may be a Taq enzyme.

(2) Primer preparation: dissolved in TE, the concentration of each universal primer is the same, the concentration of each CLO primer is also the same, the final concentration of each universal primer is 10 times the final concentration of a single CLO primer, for example: the final concentration of a single CLO primer 1.6pmol/μL, the final concentration of a single universal primer is 16pmol/μL, and the mixture formed is labeled as HXD-T.

(3) Probe preparation: dissolve with TE, the concentration of each Target probe is the same, and the concentration of each Beacon probe is also the same, and the final concentration of each Target probe is 2 times the final concentration of the Beacon probe, for example: Target The final concentration of a single probe is 1.6 pmol/μL, and the final concentration of a single Beacon probe is 0.8 pmol/μL. The premix formed by mixing is labeled as HXD-B.

(4) Reaction system preparation:

Table 1-1 PCR reaction system preparation information table

3. Detection principle

(1) The nucleic acid of the sample to be tested is contacted with the above-mentioned upstream and downstream primers, Target probe, Beacon probe and a DNA polymerase with 5'-3' exonuclease activity, and placed in a PCR operation system. Under the action of DNA polymerase (with 5'-3'exo-cutting activity, without 3'-5'exo-cutting activity), it extends forward, and when it encounters a Target probe that matches and binds to the template, the polymerase converts the Target probe The non-homologous partial sequence at the 5' end is digested to generate a free 5' end region.

(2) The free 5' end region of the Target probe is reversely complementary to the loop region of the Beacon probe, and continues to be extended under the action of the polymerase, so that the Beacon probe becomes a complete double strand.

(3) The Beacon probe is naturally coiled in a single-stranded state, and the reporter group (R) is close to the 4 consecutive G bases at the 5' end without fluorescence, and binds to the 5' end region of the free Target probe , when the amplification is double-stranded, the Beacon probe will fluoresce due to the distance between the reporter group and the 4 G bases. According to the results of the melting curve analysis, it is confirmed whether each target nucleotide sequence to be tested exists in the sample, and then it is determined whether the pathogen corresponding to each target nucleic acid sequence exists in the sample to be tested.

4. PCR reaction procedure

A touchdown PCR program was used, and the annealing temperature was decreased by 1 °C for each additional cycle in the first 6 cycles. The specific procedure is as follows:

Table 1-2

5. Analysis results

Take FAM, VIC, and ROX channels to detect target pathogens, and CY5 channel to detect internal references as an example:

1) In the FAM, VIC, ROX channels, when there is a melting peak within the Tm reference value range of a specific pathogen, it is determined that the pathogen is positive;

2) When two or more melting peaks appear at the same time, it is determined that the sample is infected with two or more pathogens at the same time;

3) When there is no melting peak in FAM, VIC, and ROX channels, and there is a melting peak in CY5 channel, it is determined that the sample has no pathogenic infection within the detection range;

4) If there are no melting peaks in the FAM, VIC, ROX, and CY5 channels, it is determined that the sample is invalid, and it is recommended to re-sample or re-extract the nucleic acid before testing.

The optimization of embodiment 2 multiplex nucleic acid detection system

(1) Combination screening of CLO-type primers and universal primers

Taking the detection of influenza A virus as an example, for the conserved segment of the M1 gene of influenza A virus, use the primer design software Primer Express 3.0 to determine the position of the 3' end of the CLO primer and the 3' end of the Target probe, and design a segment in the middle of the CLO primer The artificial sequence consists of 15 to 25 bases. Evaluate the Tm value (50-60°C) and GC content (40%-60%) of the primers. After the design is completed, submit the sequence to Sangon Bioengineering (Shanghai) Co., Ltd. for the synthesis of primers and probes, and screen out the high-sensitivity primers through experiments. , Primer-probe combination with good specificity.

It was found that the preferred primer-probe combinations for influenza A virus were combination 1 and combination 2, wherein the difference between combination 1 and combination 2 was that the universal primer sequences were inconsistent. Combination 1 and combination 2 were used to detect influenza A virus quality control products (Guangzhou Bangdesheng Biotechnology Co., Ltd., the same below), and the amplification curve and melting curve were compared and analyzed.

Table 2-1 Influenza A virus primer probe combination 1

Table 2-2 Influenza A virus primer probe combination 2

Combination 1 and combination 2 were used to detect influenza A virus quality control products, and the amplification curve and melting curve of the detection results are shown in Figure 2 below.

The above data show that the universal primers in combination 2 have better amplification efficiency.

(2) Landing PCR and conventional PCR procedures

Two clinical samples of influenza A virus were selected, and the same reaction system was used to amplify using touchdown PCR and common PCR procedures respectively, and the PCR products were analyzed by electrophoresis using 2% agarose gel. Table 2-3 Landing PCR program

Table 2-4 General PCR program

The results of the agarose gel electrophoresis experiment are shown in Figure 3 below. The amplified product bands using the touchdown PCR program are single, and the brightness is higher than that of the ordinary PCR program, showing that the non-specific bands are reduced.

The above data show that the use of touchdown PCR can reduce non-specific amplification, thereby improving the amplification efficiency. A preferred PCR procedure is a touchdown PCR procedure.

(3) Target probe optimization

After designing the Target probe of influenza A virus, synthesize two Target probes (T1 and T2) respectively, and the 3' ends of T1 and T2 are blocked with C3 to prevent the 3' end of the elongation stage from being carried out under the action of DNA polymerase. Extending, and T2 modifies LNA at the 5' end to increase the Tm value of the entire probe, and at a higher annealing temperature, it can hybridize with the target gene, thereby reducing non-specific amplification. In other systems with the same conditions, two Target probes (T1 and T2) were used to detect influenza A virus. The pros and cons of Target probes (T1 and T2) were evaluated by analyzing the Ct value of the amplification curve and the melting peak height of the melting curve. The sequences of T1 and T2 are shown in the table below:

Table 2-5 Target probe sequence

The experimental results are as shown in Figure 4. The Ct value of the amplification curve of the T2 probe is smaller than that of the T1 probe, indicating that its amplification efficiency is better than that of T1, and the melting peak height in the melting curve of the T2 probe is higher than that of T1.

The experimental results show that after the Target probe is modified with LNA at the 5', the amplification efficiency can be improved, the non-specific amplification can be reduced, and the melting peak height can be increased.

(4) Beacon probe optimization

For influenza A virus, the Beacon probe (B1) modified by the quenching group (BHQ1) and the Beacon probe (B2) quenched by four consecutive G bases were respectively designed. The quality of Beacon probes (B1 and B2) was evaluated by analyzing the Ct value of the amplification curve and the melting peak height of the melting curve.

Table 2-6 Beacon probes

The experimental results are shown in Figure 5 below. The Ct value and plateau phase of the amplification curve of the B1 probe and the B2 probe detecting influenza A virus are consistent, and the Tm value and peak height of the melting curve are also consistent, indicating that there is almost no difference between the two.

The experimental results show that the fluorescence quenching function can be realized by using 4 consecutive G bases, which is equivalent to the fluorescence quenching group. At the same time, the cost of probe synthesis can be reduced, thereby reducing medical costs and reducing the burden on patients.

The composition of embodiment 3 respiratory pathogen nucleic acid detection kits

1. Primers and probes

The nucleic acid detection kit for respiratory pathogens of the present invention includes an amplification primer set and a detection probe set for the nucleic acid sequence of respiratory pathogens, wherein the amplification primers in the kit are specifically shown in Table 3-1 below ("F" represents an upstream primer, "R" indicates a downstream primer), and the probes for each respiratory pathogen are shown in Table 3-2 below:

Table 3-1

Table 3-2

The "-" connection in the table indicates the position of the modifying group and the name of the modifying group.

2. Quality Control

The kit contains a negative quality control and a positive quality control, and the negative and positive quality controls and the samples to be tested need to be processed synchronously. Positive quality control products consist of pseudoviruses containing influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae, parainfluenza virus and internal reference gene fragments; negative quality control The product consists of a pseudovirus containing internal reference gene fragments. Pseudoviruses were purchased from Fubaiao (Suzhou) Biotechnology Co., Ltd. and prepared.

3. PCR system

3.1 Primer preparation: CLO-F1/CLO-R1～CLO-F12/CLO-R12 and Up-F, Up-R, a total of 26; centrifuge at 10,000rpm for 3min; dissolve with TE to 100pmol/μL mother solution, according to the table below 3-3 Preparation of premix:

Table 3-3

The 100 μL premix prepared according to the above table, the final concentration of CLO-F1/CLO-R1~CLO-F12/CLO-R12 is 1.6 pmol/μL, and the final concentration of Up-F and Up-R is 16 pmol/μL, marked as HXD -T.

3.2 Probe preparation: T1/B1～T11/B12, 21 in total; centrifuge at 10,000rpm for 3min; dissolve with TE to 100pmol/μL mother solution, prepare premix according to the following table 3-4:

Table 3-4

序号serial number	组分components	添加体积(μL)Add volume (μL)
11	T1～T12，12条T1～T12, 12 pieces	1.61.6
22	B1～B12，9条B1～B12, 9 pieces	0.80.8
33	TE溶液TE solution	73.673.6

Prepare 100 μL premix according to the above table, the final concentration of T1~T12 single probe is 1.6 pmol/μL, the final concentration of B1~B12 single probe is 0.8 pmol/μL, and the premix is marked as HXD-B.

3.3 Referring to Example 1, the PCR reaction system was prepared. The nucleic acids were the sample to be tested, the negative control, and the positive control, respectively, oscillated and mixed, and centrifuged before loading on the machine.

4. PCR reaction procedure

Referring to Example 1, the PCR reaction program setting was performed.

5. Result analysis

The Tm value ranges of the eight respiratory pathogens in each channel are shown in Table 3-5 below:

Table 3-5

Positive control interpretation: There should be melting peaks in FAM, VIC, ROX and CY5 channels, corresponding to influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae, The melting peaks of parainfluenza virus and internal reference gene determine that the positive quality control product is qualified. If one or more of them has no melting peak, it is determined that the reagent is invalid; if there are melting peaks other than pathogens, it is determined that there is contamination in the current test.

Wherein the qualified positive quality control product adopts the detection result of the kit of the present invention as shown in Figure 6 and Figure 7.

Negative control interpretation: There is no melting peak in the FAM, VIC, and ROX channels, and there is a melting peak in the CY5 channel, which corresponds to the melting peak of the internal reference gene, and the negative quality control product is determined to be qualified. If there are melting peaks in the FAM, VIC, and ROX channels, it is determined that there is contamination in the current detection.

For example, some test results are shown in Table 3-6 below, and the corresponding analysis of the test results is as follows:

Table 3-6

Note: + indicates that there is a melting peak, - indicates that there is no melting peak, ± indicates that there is or is not melting peak, / indicates that there is no detection target at this Tm value.

Using the above-mentioned nucleic acid detection kit for respiratory pathogens, according to the above-mentioned method steps, the collected clinical specimens of throat swabs were extracted for nucleic acid detection, and the detection results were statistically shown in Tables 3-7-3-13 below. The control method was the detection of 13 kinds of respiratory pathogens multiplex detection kit (PCR capillary electrophoresis fragment analysis method) (National Machinery Injection No. 20183400518).

Table 3-7 Influenza A negative and positive statistical table

Table 3-8 Statistical Table of Influenza B Virus Negative and Positive

Table 3-9 Statistical Table of Respiratory Syncytial Virus Negative and Positive

Table 3-10 Statistical Table of Rhinovirus Positive and Positive

Table 3-11 Statistical Table of Adenovirus Negative and Positive

Table 3-12 Statistical table of negative and positive results of human metapneumovirus

Table 3-13 Parainfluenza virus negative and positive statistical table

Table 3-14 Mycoplasma pneumoniae positive and negative statistical table

The above statistics show that the respiratory pathogen nucleic acid detection kit of the present invention has excellent performance for clinical influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae, and parainfluenza virus samples. detection performance.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims

A multiplex nucleic acid detection system, characterized in that it includes an amplification primer set and a detection probe set for a target nucleic acid sequence, and the detection probe set includes a Target probe and a Beacon probe,

The composition of the Target probe from the 5' end to the 3' end is as follows: 5' end region, Target region, 3' end region;

The composition of the Beacon probe from the 5' end to the 3' end is as follows: 5' end region, loop region, and 3' end region;

The 5' end of the 5' end region sequence of the Target probe is modified with LNA, the 3' end of the 3' end region sequence is modified with C3, and the Target region sequence can be reverse complementary to the target nucleic acid sequence;

The 5' end of the 5' end region sequence of the Beacon probe is n consecutive guanines, n is an integer of 1-8, the 3' end of the 3' end region sequence is modified with a fluorescent reporter group, and the loop The region sequence is reverse complementary to the 5' region sequence.
The multiple nucleic acid detection system according to claim 1, wherein the 3' end of the 3' end region sequence of the Target probe is CGCG.
The multiple nucleic acid detection system according to claim 1, wherein the sequence length of the 5' end region of the Beacon probe is 5-8 bp, the sequence length of the 3' end region is 5-8 bp, and/or the The sequence length of the loop region is 30-50 bp.
The multiplex nucleic acid detection system according to any one of claims 1-3, wherein the number n of continuous guanines at the 5' end of the 5" end region sequence of the Beacon probe is an integer of 3-5.
The multiplex nucleic acid detection system according to any one of claims 1-4, wherein the set of amplification primers comprises a pair of CLO primers,

The composition of each CLO primer in the CLO primer pair from the 5' end to the 3' end is as follows: 5' end region, loop region, and 3' end region;

The sequence length of the 5' end region is 18-25 bp, the sequence length of the 3' end region is 10-15 bp, and/or the sequence length of the loop region is 15-25 bp.
The multiple nucleic acid detection system according to claim 5, wherein the amplification primer set further comprises a pair of universal primers, and the upstream universal primer in the universal primer pair is consistent with the loop region of the upstream CLO primer in the CLO primer pair; The downstream universal primer in the universal primer pair is consistent with the loop region of the downstream CLO primer in the CLO primer pair.
According to the multiplex nucleic acid detection system described in any one of claims 1-6, it is characterized in that, the 3' terminal modified fluorescent reporter group of the 3' end region sequence of the Beacon probe is selected from FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas, Red, LC RED640, Cy5, LC RED705, Alexa Fluor 488, and Alexa Fluor 750.
A multiplex nucleic acid detection method, is characterized in that, comprises the steps:

Obtain the nucleic acid of the biological sample to be tested;

The nucleic acid of the biological sample, the DNA polymerase and the multiple nucleic acid detection system described in any one of claims 1-7 are mixed to form a PCR reaction system, and the PCR reaction and melting curve analysis are carried out.
The multiplex nucleic acid detection method according to claim 8, characterized in that the reaction procedure of the PCR reaction is touch down PCR.
According to the multiple nucleic acid detection method according to any one of claims 8-9, it is characterized in that, in the PCR reaction system, the concentration of each CLO primer for different target nucleic acid sequences is the same, and the concentration of each universal primer is also the same, and The final concentration of each universal primer is 5-15 times that of the CLO single primer.
The multiplex nucleic acid detection method according to claim 10, characterized in that the final concentration of each universal primer is 9-11 times the final concentration of a CLO single primer.
According to the multiple nucleic acid detection method described in any one of claims 8-9, it is characterized in that, in the PCR reaction system, the concentration of each Target probe for different target nucleic acid sequences is the same, and the concentration of each Beacon probe is the same, The final concentration of each Target probe was 1-5 times the final concentration of the Beacon probe.
The multiplex nucleic acid detection method according to claim 12, wherein the final concentration of each Target probe is twice the final concentration of the Beacon probe.
The multiplex nucleic acid detection method according to any one of claims 8-9, wherein the method can detect 1 to 20 types of target nucleic acid sequences.