CN114134219A

CN114134219A - Multiple nucleic acid detection system and preparation method and application thereof

Info

Publication number: CN114134219A
Application number: CN202111537083.4A
Authority: CN
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: Guangzhou Jinqirui Biotechnology Co ltd
Current assignee: Guangzhou Jinqirui Biotechnology Co ltd
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2022-03-04
Also published as: WO2023109032A1

Abstract

The invention relates to a multiple nucleic acid detection system, which comprises an amplification primer group and a detection probe group aiming at a Target nucleic acid sequence, wherein the detection system comprises a Target probe modified by LNA and a Beacon probe achieving a fluorescence quenching effect through 1-8 continuous G bases, and based on the proposal of the multiple nucleic acid detection system, the inventor combines a touchdown PCR program, and also provides a multiple nucleic acid detection method, which can further reduce non-specific amplification in PCR reaction and improve detection sensitivity. Therefore, the method overcomes the limitation of the traditional real-time fluorescent quantitative PCR typing, realizes single-tube multiple typing through analysis of special signals and melting curves, and realizes accurate qualitative detection on each target gene in at most 20 target nucleic acid sequences to be detected in a sample by a simpler reaction system and lower detection cost.

Description

Multiple nucleic acid detection system and preparation method and application thereof

Technical Field

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

Background

The real-time fluorescent quantitative PCR method is a nucleic acid detection method commonly used in molecular biology, and compared with a common PCR method, the method is simple and convenient to operate and wide in application. In the process of carrying out PCR amplification on a target gene of a sample to be detected, a fluorescence signal generated by a reaction system is monitored in real time through an instrument, and the PCR process is detected in real time. The common PCR amplification technology needs to perform electrophoresis analysis on products after amplification is completed, the analysis process is complicated and time-consuming, and meanwhile, the uncovering of PCR products can cause pollution to the laboratory environment.

The method for realizing the fluorescent signal is mainly divided into a probe method and a dye method, wherein the probe method is used for monitoring the fluorescent signal released by a probe in real time by adding an oligonucleotide probe marked by a fluorescent group into a reaction system so as to realize the specific detection of a target sequence; the dye method is that double-stranded DNA fluorescent dye is added into a reaction system, and the fluorescent dye is combined into a DNA double-stranded minor groove to realize the detection of a target sequence;

in the real-time fluorescent PCR detection mode, PCR amplification and detection of the target sequence are performed simultaneously without additional steps. Therefore, the real-time detection mode is simple and direct. However, the maximum number of target sequences that can be detected in a single-tube assay in this mode is limited by the number of fluorescence detection channels of the real-time PCR instrument, and generally does not exceed 6. Therefore, based on the direct and simple methodological advantages, the real-time fluorescent quantitative PCR method needs to be improved in order to realize the detection of more target gene number in the single-tube detection.

US 2013/0109588 a1 discloses a real-time fluorescent PCR assay method useful for melting curve analysis, which achieves detection of a target sequence by designing two probes (PTO probe and CTO probe). When the method described in this patent application is used to perform multiplex real-time PCR that distinguishes each target sequence, one PTO probe and one CTO probe need to be designed separately for each target sequence, i.e. 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, using 2 PTO probes and 2 CTO probes. In this case, the method of this patent is more complicated and expensive than the conventional multiplex PCR using a single fluorescent probe for each target sequence.

US 2015/0072887 a1 discloses a real-time PCR assay useful for melting curve analysis, which enables real-time detection of target sequences by 3 probes. However, when the method described in this patent application is used to perform multiplex real-time PCR requiring discrimination of each target sequence, 3 probes need to be designed for each target sequence separately, which results in more complicated reaction system and high detection cost.

Disclosure of 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.

The technical scheme is as follows:

the multiple nucleic acid detection system is characterized by comprising an amplification primer group and a detection probe group aiming at a Target nucleic acid sequence, wherein the detection probe group comprises a Target probe and a Beacon probe, and the Target probe sequentially comprises the following components from a 5 'end to a 3' end: 5 'end region, Target region and 3' end region; the Beacon probe sequentially comprises the following components from a 5 'end to a 3' end: a 5 'end region, a loop region, a 3' end region; LNA is modified at the 5 'end of the 5' end region sequence of the Target probe, C3 is modified at the 3 'end of the 3' end region sequence, and the Target region sequence can be reversely complementary with a Target nucleic acid sequence; the 5 'end of the 5' end region sequence of the Beacon probe is n continuous guanines, n is an integer from 1 to 8, the 3 'end of the 3' end region sequence is modified with a fluorescent reporter group, and the 3 'end region sequence is reversely complementary with the 5' end region sequence.

It is also an object of the present invention to provide a multiplex nucleic acid detection method.

The technical scheme is as follows:

obtaining nucleic acid of a biological sample to be detected;

mixing the biological sample nucleic acid, DNA polymerase and the multiple nucleic acid detection system to prepare a PCR reaction system, and carrying out PCR reaction and melting curve analysis.

The inventor of the invention develops a more convenient and efficient multiple nucleic acid detection system based on the intensive research on a gene detection technology, the detection system comprises a dual-probe system of a Target probe and a Beacon probe, and the inventor finds out through experiments that through the structural modification of the Target probe and the Beacon probe, particularly, a 5 'end region and a 3' end region in the Target probe are designed into non-homologous sequences and are not combined with a template, the 5 'end region modifies LNA (low-noise amplifier), the Tm value of the Target probe is improved, the 3' end region provides a CGCG base chain, and then C3 is modified for sealing to block non-specific extension; the intermediate Target region may be reverse complementary to the Target nucleic acid sequence, effectively providing a binding sequence anchor. Meanwhile, the sequence of the 5 'end region of the Target probe is reversely complementary with the loop region of the Beacon probe, the Beacon probe is naturally coiled in a free state, the 5' end region is cut off in a free state under the action of 5 '→ 3' exonuclease activity of DNA polymerase during Target nucleic acid detection because the reporter group (R) is close to a plurality of (1-8) G bases continuous to the 5 end, the Beacon probe can be hybridized and combined with the loop region of the Beacon probe at the temperature lower than the Tm value, the Beacon probe extends under the action of 5 '→ 3' polymerase activity of the DNA polymerase and is amplified into a double strand, and the Beacon probe fluoresces because the reporter group is far away from the plurality of G (1-8) bases. Thereby effectively reducing the synthesis cost of the probe and improving the detection sensitivity, specificity and stability while non-specific amplification in the detection reaction.

And the detection system eliminates the difference of different amplicons in the amplification products caused by the difference of the amplification efficiency between the specific primers by the combined use of the CLO primers and the universal primers. Thereby more stably realizing the sample detection as a whole.

Based on the proposal of the multiple nucleic acid detection system, the inventor combines the design of a PCR program, designs the PCR program and combines the use of a touchdown PCR program, and also provides a multiple nucleic acid detection method, which can further reduce the non-specific amplification in the PCR reaction and improve the detection sensitivity. Therefore, the limitation of the traditional real-time fluorescence quantitative PCR typing is overcome, single-tube multiple typing is realized through analysis of special signals and melting curves, accurate qualitative detection can be realized for each target gene in at most 20 target nucleic acid sequences to be detected in a sample to be detected in a real-time fluorescence quantitative PCR instrument by using a simpler reaction system and lower detection cost, and the specificity of detection can be ensured by judging and reading a specific melting peak in the melting curve.

Drawings

FIG. 1 is a schematic diagram showing the design of each component of the multiplex nucleic acid detecting system of the present invention.

FIG. 2 is a graph comparing the results of detection of influenza A virus using different primer probe combinations in example 2.

FIG. 3 is an agarose gel electrophoresis of the influenza A PCR product amplified using the different PCR procedures of example 2.

FIG. 4 is a graph comparing the results of influenza A virus detection using different Target probes in example 2.

FIG. 5 is a graph comparing the results of influenza A virus detection using different Beacon probes in example 2.

Fig. 6 is a partial result of the detection of the positive quality control material by using the respiratory tract pathogen typing detection kit in example 3, wherein a is a melting curve chart under the FAM channel, C is an amplification curve chart under the FAM channel, B is a melting curve chart under the VIC channel, and D is an amplification curve chart under the VIC channel.

FIG. 7 is a partial result of the positive quality control test using the kit for testing respiratory tract pathogens in example 3, wherein A is a melting profile under ROX channel, C is an amplification profile under ROX channel, B is a melting profile under CY5 channel, and D is an amplification profile under CY5 channel.

Detailed Description

Experimental procedures without specific conditions noted in the following examples, generally followed by 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 manufacturer's recommendations. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Furthermore, as used herein, the term "or" is an inclusive "or" symbol and is equivalent to the term "and/or," unless the context clearly dictates otherwise.

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

The present invention will be described in further detail with reference to specific examples.

Some embodiments of the present invention provide a multiplex nucleic acid detection system, including an amplification primer set and a detection probe set for a Target nucleic acid sequence, where the detection probe set includes a Target probe and a Beacon probe, and the Target probe sequentially includes, from 5 'end to 3' end: 5 'end region, Target region and 3' end region; the Beacon probe sequentially comprises the following components from a 5 'end to a 3' end: a 5 'end region, a loop region, a 3' end region; LNA is modified at the 5 'end of the 5' end region sequence of the Target probe, C3 is modified at the 3 'end of the 3' end region sequence, and the Target region sequence can be reversely complementary with a Target nucleic acid sequence; the 5 ' end of the 5 ' end region sequence of the Beacon probe is n continuous 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 region sequence is reversely complementary with the 5 ' end region sequence of the Target probe.

In some of these embodiments, the set of detection probes comprises Target probes and Beacon probes for 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, 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 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 non-specific amplification during detection can be reduced.

In some embodiments, the 5 'end region and the 3' end region in the Target probe are non-homologous sequences and are not combined with a template, the 5 'end region modifies LNA (low noise amplifier), the Tm value of the Target probe is increased, the 3' end region provides a CGCG base chain, and C3 is further modified for blocking non-specific extension; the intermediate Target region may be reverse complementary to the Target nucleic acid sequence, effectively providing a binding sequence anchor.

In some embodiments, the length of the 5 ' terminal region sequence of the Beacon probe is 5-8 bp, the length of the 3 ' terminal region sequence is 5-8 bp, and/or the length of the loop region sequence is 30-50 bp, and the loop region sequence is reversely complementary with the 5 ' terminal region sequence of the Target probe.

In some embodiments, the number n of consecutive guanines at the 5 'end of the sequence in the 5' end region of the above Beacon probe is an integer of 3 to 5. More preferably, n is 4, thereby effectively providing a fluorescence quenching function.

In some embodiments, the amplification primer set comprises a CLO primer pair, and each CLO primer in the CLO primer pair sequentially comprises, from 5 'end to 3' end: a 5 'end region, a loop region, a 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 the sequence length of the loop region is 15-25 bp.

In some embodiments, the amplification primer set comprises CLO primer pairs for 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, 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 nucleic acid sequences of interest.

In some embodiments, the amplification primer set further comprises a universal primer pair, wherein the upstream primer in the universal primer pair is identical to the loop region of the upstream CLO primer in the CLO primer pair; and the downstream primer in the universal primer pair is consistent with the loop region of the downstream CLO primer in the CLO primer pair.

In some embodiments, the universal primers can amplify all PCR products generated from the first 2 cycles after the 3 rd cycle of PCR amplification, thereby ensuring amplification uniformity of each target.

In some embodiments, in the multiplex nucleic acid detection system, the 3 '-end modified fluorescent reporter group in the sequence at the 3' -end region of the Beacon probe is selected from the group consisting of 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 invention also provide application of the multiplex nucleic acid detection system in preparing a reagent or a kit for disease diagnosis.

In some embodiments, the disease is a cross-infectious disease caused by multiple pathogens. Preferably, the infectious disease is a respiratory tract infection, a digestive tract infection, a blood infection, a urinary tract infection, or the like. Further preferably, the kit is used for detecting and determining pathogens in respiratory tract infection with similar symptoms such as cough, and can be influenza virus such as influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae, parainfluenza virus and the like.

Some embodiments of the invention also provide a multiplex nucleic acid detection method comprising the steps of:

obtaining nucleic acid of a biological sample to be detected;

mixing the biological sample nucleic acid, DNA polymerase and the multiple nucleic acid detection system of any one of claims 1-7 to prepare a PCR reaction system, and performing PCR reaction and melting curve analysis.

In some embodiments, the melting curve analysis is performed on the product obtained from the PCR reaction to determine the type of the target nucleic acid sequence present in the reaction system, thereby implementing the multiplex nucleic acid detection.

In some of these embodiments, the reaction sequence of the PCR reaction is touchdown PCR. More preferably, when the touchdown PCR procedure is used for detection, the annealing temperature is decreased by 1 ℃ for each 1 cycle of the previous 6 cycles.

In some embodiments, the biological sample may be selected from the group consisting of, 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, artificially synthesized plasmid samples.

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

In some embodiments, in the PCR reaction system, the concentrations of CLO primers and universal primers are the same, and the final concentration of each universal primer is 5-15 times, and more preferably 10 times, the final concentration of the CLO primer is the same as the final concentration of a single CLO primer.

In some embodiments, in the PCR reaction system, the concentration of each Target probe for different Target nucleic acid sequences is the same, the concentration of each Beacon probe is the same, and the final concentration of each Target probe is 1 to 5 times, and more preferably 2 times, the final concentration of each Beacon probe.

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

In some embodiments, the methods described above are not limited by theory, and the resolution or accuracy of the melting curve analysis can be as high as 0.5 ℃ or higher. In other words, melting curve analysis can distinguish two melting peaks having melting points that differ by only 0.5 ℃ or less (e.g., 0.1 ℃, 0.2 ℃, 0.3 ℃, 0.4 ℃, 0.5 ℃).

In some embodiments, the difference in melting point between the amplification products of any two test target nucleic acid sequences detectable by the above methods can be at least 0.5 ℃, such that the any two test target nucleic acid sequences can be distinguished and distinguished by melting curve analysis. However, for the purpose of facilitating differentiation and discrimination, a larger difference in melting point between any two target nucleic acid sequences to be detected is preferable in some cases.

In some embodiments, the melting point difference detectable by the above methods between any two target nucleic acid sequences to be detected can be any desired value (e.g., at least 0.5 ℃, at least 1 ℃, at least 2 ℃, at least 3 ℃, at least 4 ℃, at least 5 ℃, at least 8 ℃, at least 10 ℃, at least 15 ℃, or at least 20 ℃) as long as the melting point difference can be distinguished and distinguished by melting curve analysis.

Some embodiments of the invention also provide an influenza a virus detection kit, which comprises the above multiple nucleic acid detection system aiming at the conserved segment sequence of the influenza a virus M1 gene.

In some embodiments, the multiplex nucleic acid detection system comprises an amplification primer group and a detection probe group, wherein in the amplification primer group, the sequence of a CLO primer pair is shown as SEQ ID NO. 1-SEQ ID NO.2 or shown as SEQ ID NO. 7-SEQ ID NO. 8; the general primer pair is shown as SEQ ID NO. 5-SEQ ID NO.6 or SEQ ID NO. 9-SEQ ID NO. 10; in the detection probe group, the Target probe sequence is shown as SEQ ID NO. 3; the sequence of the Beacon probe is shown as SEQ ID NO.4 or SEQ ID NO. 11. Furthermore, the sequence of the CLO primer pair is shown as SEQ ID NO. 7-SEQ ID NO. 8; the general primer pair is shown as SEQ ID NO. 9-SEQ ID NO. 10; in the detection probe group, the Target probe sequence is shown as SEQ ID NO. 3; the sequence of the Beacon probe is shown in SEQ ID NO. 4.

EXAMPLE 1 composition of multiplex nucleic acid detection System

1. Primer probe

Aiming at an object to be detected, referring to related professional documents, determining a nucleic acid sequence conserved segment of the object to be detected according to literature research, selecting at least 1 segment of specific Target gene sequence (Target nucleic acid sequence), and designing an upstream oligonucleotide primer CLO-F (Convex loop oligo-Forward, CLO-F), a downstream oligonucleotide primer CLO-R (Convex loop oligo-Reverse, CLO-R), a Target probe (T probe) sequence Target gene region, an artificial sequence Beacon probe (B probe) and a universal primer based on the selected specific Target gene sequence, wherein CLO-F comprises a sequence complementary with the specific Target gene sequence, CLO-R comprises a sequence identical with the specific Target gene sequence, and the Target probe is divided into three parts, a 5 end region, a Target gene region and a 3 end region according to turning points at two ends complementary with the specific Target sequence, the Target gene region of the Target probe is complementary with a specific Target sequence, the 5 end and the 3 end regions are artificially introduced sequences, and the 5 end regions of all the Target probes are different from each other in sequence. As described below in detail in connection with fig. 1.

CLO primer: the total of the components is 3. The 5' end anchoring section consists of 18-25 bases and has a higher Tm value; the 3' end is a specific binding region which consists of 10-15 basic groups and has a lower Tm value; the middle loop area is composed of 15-25 basic groups of an artificial sequence.

Target probe: consists of 3 parts. The 5 'end and the 3' end are non-homologous sequences, are not combined with a template, 5 'modifies LNA, improves the Tm value of a Target probe, 3' provides a CGCG base chain, and then modifies C3 for sealing; the intermediate target gene region and the template are reverse complementary sequences, providing an anchor point for the binding sequence.

Beacon probe: and providing a fluorescence signal release system to realize melting curve analysis. Consists of 3 parts. The 5' end consists of 5-8 bases, and the tail end is provided with 4 continuous G bases to provide a fluorescence quenching function; the loop region consists of 30-50 bases and is reversely complementary with the artificial sequence at the 5' end of the Target probe; the 3' end consists of 5-8 basic groups, and the tail end is modified with a fluorescent reporter group. Aiming at different target nucleic acid sequences, the designed Beacon probe has different end-modified fluorescent reporter groups.

The general primer is as follows: the sequence and the structure are consistent with the Loop region of the CLO primer, and after the 3 rd cycle of PCR amplification is performed, all PCR products generated in the first 2 cycles can be amplified, so that the amplification uniformity of each target is ensured.

2. The system formulation and composition (for example, the RNA in a biological sample, for example, the DNA in a biological sample, can be removed by removing the components and reaction procedures related to reverse transcription as follows)

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

(2) Preparing a primer: dissolving with TE, wherein the concentration of each universal primer is the same, the concentration of each CLO primer is the same, and the final concentration of each universal primer is 10 times of that of a CLO single primer, for example: the final concentration of the CLO primer single strand was 1.6 pmol/. mu.L, the final concentration of the universal primer single strand was 16 pmol/. mu.L, and the label formed by mixing was HXD-T.

(3) Preparing a probe: dissolving with TE, wherein the concentration of each Target probe is the same, the concentration of each Beacon probe is also the same, and the final concentration of each Target probe is 2 times of that of the Beacon probe, for example: the final concentration of the single Target probe strip is 1.6 pmol/. mu.L, the final concentration of the single Beacon probe strip is 0.8 pmol/. mu.L, and the mixed premixed solution is marked as HXD-B.

(4) Preparing a reaction system:

TABLE 1-1 information table for PCR reaction system preparation

3. Principle of detection

(1) And (2) contacting the nucleic acid of a sample to be detected with the upstream and downstream primers, the Target probe and the Beacon probe and DNA polymerase with 5 '-3' exonuclease activity, placing the nucleic acid in a PCR running system, extending the CLO primer forwards under the action of the DNA polymerase (with 5 '-3' exonuclease activity and without 3 '-5' exonuclease activity), and when encountering the Target probe matched and combined with the template, carrying out enzyme digestion on a non-homologous part sequence at the 5 'end of the Target probe by the polymerase to generate a free 5' end region.

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

(3) The Beacon probe is naturally curled in a single-stranded state, the distance between the reporter group (R) and 4 continuous G bases at the 5 'end is short, no fluorescence exists, and when the Beacon probe is combined with the 5' end region of a free Target probe and amplified into a double strand, the Beacon probe fluoresces because the distance between the reporter group and 4G bases is long. From the results of the melting curve analysis, it is confirmed whether or not each target nucleic acid sequence to be detected is present in the sample, and further, whether or not the pathogen corresponding to each target nucleic acid sequence is present in the sample to be detected is determined.

4. PCR reaction procedure

The touchdown PCR procedure was used, 6 cycles earlier, with 1 cycle increase, and 1 ℃ decrease in annealing temperature. The specific procedure is as follows:

tables 1 to 2

5. Analysis results

Taking FAM, VIC and ROX channels to detect target pathogens, and taking CY5 channel detection internal reference as an example:

1) in FAM, VIC and ROX channels, when a melting peak exists in a specific pathogen Tm reference value range, the pathogen is judged to be positive;

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

3) when FAM, VIC and ROX channels have no melting peak and CY5 channel has a melting peak, judging that the sample has no pathogen infection in the detection range;

4) if no melting peak exists in FAM, VIC, ROX and CY5 channels, the sample is judged to be invalid, and resampling or nucleic acid re-extraction and then detection are recommended.

Example 2 optimization of multiplex nucleic acid detection System

(1) Combinatorial screening of CLO-type and Universal primers

Taking detection of influenza A virus as an example, for M1 gene conserved segments of influenza A virus, Primer design software Primer Express 3.0 is used for determining the positions of the 3 'end of a CLO Primer and the 3' position of a Target probe, and the CLO Primer is designed to be composed of 15-25 basic groups of an artificial sequence in the middle. And (3) evaluating the Tm value (50-60 ℃) and the GC content (40-60%) of the primer, submitting the sequence to a biological engineering (Shanghai) corporation for primer probe synthesis after the design is finished, and screening out a primer probe combination with high sensitivity and good specificity through experiments.

Preferred primer probe sets for influenza a virus were found to be combination 1 and combination 2, where combination 1 and combination 2 differ in the universal primer sequences being non-identical. The influenza A virus quality control (Guangzhou, Bangdong Biotechnology Co., Ltd., the same below) was tested using combination 1 and combination 2, respectively, and the amplification curve and the melting curve were analyzed by comparison.

TABLE 2-1 influenza A primer Probe combination 1

TABLE 2-2 influenza A primer Probe combination 2

The influenza a virus quality control was tested using combination 1 and combination 2, respectively, and the amplification curve and melting curve of the test results are shown in fig. 2 below.

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

(2) Touchdown PCR and conventional PCR procedures

2 clinical samples of influenza a virus were selected, amplified using the same reaction system using touchdown PCR and general PCR procedures, respectively, and the PCR products were analyzed by electrophoresis using 2% agarose gel.

TABLE 2-3 touchdown PCR procedure

TABLE 2-4 general PCR procedure

The results of the agarose gel electrophoresis experiments are shown in FIG. 3 below, and the amplified products using the touchdown PCR procedure were single-banded and brighter than the normal PCR procedure, showing a reduction in non-specific bands.

The above data indicate that the use of touchdown PCR can reduce non-specific amplification and thus increase amplification efficiency. The preferred PCR procedure is the touchdown PCR procedure.

(3) Target probe optimization

After designing the Target probes of the influenza A virus, respectively synthesizing 2 Target probes (T1 and T2), wherein the 3 ' ends of T1 and T2 are both blocked by C3, so that the 3 ' ends in the extension stage are prevented from extending under the action of DNA polymerase, and the T2 modifies LNA at the 5 ' end, so that the Tm value of the whole probe is improved, and the Target probes can be hybridized at a higher annealing temperature, so that the non-specific amplification is reduced. In the system with the same conditions, 2 Target probes (T1 and T2) were used to detect influenza A virus. The advantages and disadvantages of the Target probes (T1 and T2) are 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 following table:

TABLE 2-5 Target Probe sequences

The experimental result is shown in the following figure 4, the Ct value of the amplification curve of the T2 probe is smaller than that of the T1 probe, which indicates that the amplification efficiency is better than that of T1, and the melting peak in the melting curve of the T2 probe is higher than that of T1.

Experimental results show that after LNA is added to the 5' of the Target probe for modification, the amplification efficiency can be improved, nonspecific amplification is reduced, and the melting peak height is improved.

(4) Beacon probe optimization

A Beacon probe (B1) modified by a quenching group (BHQ1) and a Beacon probe (B2) quenched by 4 continuous G bases are respectively designed for influenza A viruses, the influenza A viruses are detected in a system with the same other conditions, and the advantages and the disadvantages of the Beacon probes (B1 and B2) are evaluated by analyzing the Ct value of an amplification curve and the melting peak height of a melting curve.

TABLE 2-6 Beacon probes

As shown in FIG. 5, the Ct value and the plateau phase of the amplification curve of influenza A virus detected by the B1 probe and B2 probe were identical, and the Tm value and the peak height of the melting curve were identical, indicating that there was almost no difference between the two.

The experimental result shows that the fluorescent quenching function can be realized by using 4 continuous G bases, and the fluorescent quenching function is equivalent to a fluorescent quenching group. Meanwhile, the synthesis cost of the probe can be reduced, so that the medical cost is reduced, and the burden of a patient is relieved.

EXAMPLE 3 composition of nucleic acid detection kit for respiratory pathogens

1. Primers and probes

The respiratory tract pathogen nucleic acid detection kit comprises an amplification primer group and a detection probe group aiming at a nucleic acid sequence of a respiratory tract pathogen, wherein the amplification primers in the kit are specifically shown in the following table 3-1 (the 'F' represents an upstream primer, and the 'R' represents a downstream primer), and the probes aiming at the respiratory tract pathogens are specifically shown in the following table 3-2:

TABLE 3-1

TABLE 3-2

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

2. Quality control product

The kit contains a negative quality control product and a positive quality control product, and the negative and positive quality control products and a sample to be detected need to be synchronously processed. The positive quality control product consists of pseudoviruses containing influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae, parainfluenza virus and internal reference gene segments; the negative quality control product consists of pseudovirus containing reference gene segments. Pseudoviruses were purchased from Bai' ao (Suzhou) Biotech limited.

3. PCR system

3.1 primer preparation: CLO-F1/CLO-R1-CLO-F12/CLO-R12 and Up-F, Up-R, for 26 strips; centrifuging at 10,000rpm for 3 min; dissolve with TE to 100 pmol/. mu.L stock solution, prepare premix according to the following tables 3-3:

tables 3 to 3

100 μ L of the premix prepared in the above table had a final single CLO-F1/CLO-R1-CLO-F12/CLO-R12 concentration of 1.6 pmol/. mu.L and a final single Up-F, Up-R concentration of 16 pmol/. mu.L, and was designated HXD-T.

3.2 preparing a probe: 21T 1/B1-T11/B12 in total; centrifuging at 10000rpm for 3 min; dissolve with TE to 100 pmol/. mu.L stock solution, prepare premix according to the following tables 3-4:

tables 3 to 4

Serial number	Components	Add volume (μ L)
			1	12 pieces of T1-T12	1.6
2	B1-B12, 9 strips	0.8
			3	TE solution	73.6

The 100. mu.L premix prepared in the above manner had a final concentration of 1.6pmol/μ L, B1-B12 for each probe T1-T12 and 0.8pmol/μ L for each probe, and the premix was designated HXD-B.

3.3 reference example 1, PCR reaction system preparation is carried out, nucleic acids are respectively a sample to be detected, a negative control and a positive control, the mixture is shaken and mixed evenly, and the mixture is put on a machine after centrifugation.

4. PCR reaction procedure

The PCR reaction program was set up with reference to example 1.

5. Analysis of results

The Tm values for the 8 respiratory pathogens in each channel are shown in tables 3-5 below:

tables 3 to 5

Positive control interpretation: melting peaks are found in FAM, VIC, ROX and CY5 channels, which respectively correspond to the melting peaks of influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae, parainfluenza virus and internal reference genes, and the positive quality control products are judged to be qualified. If one or more of the melting peaks do not exist, judging that the reagent is invalid; and if the melting peak except the pathogen appears, judging that the pollution exists in the current detection.

Wherein the detection results of qualified positive quality control products adopting the kit are shown in fig. 6 and 7.

Negative control interpretation: no melting peak exists in FAM, VIC and ROX channels, a melting peak exists in CY5 channel, which corresponds to the melting peak of the internal reference gene, and the negative quality control product is judged to be qualified. If the FAM, VIC and ROX channels have melting peaks, judging that the current detection has pollution.

For example, some of the test results are shown in tables 3-6 below, and the test results are analyzed as follows:

tables 3 to 6

Note: + represents the presence of a melting peak, -represents the absence of a melting peak, ± represents the presence or absence of a melting peak, and/represents the absence of a detection target at the Tm value.

By using the respiratory tract pathogen nucleic acid detection kit, the collected throat swab clinical specimens are subjected to nucleic acid extraction and then are detected according to the steps of the method, and the detection results are counted and shown in the following tables 3-7-3-13. The control method is detection of 13 respiratory tract pathogen multiple detection kits (PCR capillary electrophoresis fragment analysis method) (national mechanical standard 20183400518).

TABLE 3-7 statistical tables for negative and positive influenza A virus

TABLE 3-8 statistical tables for negative and positive influenza B virus

TABLE 3-9 statistical tables for negative and positive respiratory syncytial virus

TABLE 3-10 statistical tables for negative and positive rhinovirus

TABLE 3-11 statistical tables for negative and positive adenovirus

TABLE 3-12 statistical tables of yin-positivity of human metapneumovirus

TABLE 3-13 statistical tables of negative and positive parainfluenza viruses

TABLE 3-14 Mycoplasma pneumoniae negative and positive statistical tables

The statistical data show that the respiratory tract pathogen nucleic acid detection kit has excellent detection performance for clinical influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae and parainfluenza virus samples.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Guangzhou City gold boundary Rui Biotechnology Limited liability company

<120> multiplex nucleic acid detection system, preparation method and application thereof

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<213> Artificial Sequence

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<213> Artificial Sequence

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<212> DNA

<213> Artificial Sequence

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<212> DNA

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<212> DNA

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atgttctcta ttttgtattc ttcatctttc atatatctcg gtggtcgccg taagaacat 59

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<212> DNA

<213> Artificial Sequence

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attttggaca aagcgtctac gctgcatgta gtcatcactg agtcatcgcc tcgctcac 58

<210> 13

<211> 60

<212> DNA

<213> Artificial Sequence

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acacagatct tgaggctttc atggaatcag cagagacggc aacttatata aagacaagac 60

<210> 14

<211> 41

<212> DNA

<213> Artificial Sequence

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atcaggaaat gtagtcatca ctgagtcatc ggaacaacag c 41

<210> 15

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 15

gctgagctca gcagagacgg caacttataa tggccttc 38

<210> 16

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 16

aaacagacat aagcagctca gtaaatgtag tcatcactga gtcatcgctt ctctaggag 59

<210> 17

<211> 58

<212> DNA

<213> Artificial Sequence

<400> 17

ttcattgact tgagatattg atgcatccag cagagacggc aacttatact catcagaa 58

<210> 18

<211> 51

<212> DNA

<213> Artificial Sequence

<400> 18

cgttgccggc cgagaaggat gtagtcatca ctgagtcatc gtgcgcaggt a 51

<210> 19

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 19

gacttttgag gtggatccca tggacagcag agacggcaac ttatagccca ccctt 55

<210> 20

<211> 58

<212> DNA

<213> Artificial Sequence

<400> 20

aatacatcta cccttaccgt tacagatgta gtcatcactg agtcatcgca tgtgagct 58

<210> 21

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 21

ctaccgttaa cttctggtta aagcgctcca gcagagacgg caacttatac tcgttagca 59

<210> 22

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 22

tgtatatcaa ctgtgttcaa ctccatgtag tcatcactga gtcatcggtt gatgaaaga 59

<210> 23

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 23

cttgtctttt cttcagcaga gacggcaact tataccaagt tatc 44

<210> 24

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 24

ttctgcagct atatgtagtc atcactgagt catcgtaatc acatc 45

<210> 25

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 25

ggacatagtt cagcagagac ggcaacttat acgagcatct g 41

<210> 26

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 26

tgtatatcaa ctgtgttcaa ctccatgtag tcatcactga gtcatcggtt gatgaaaga 59

<210> 27

<211> 60

<212> DNA

<213> Artificial Sequence

<400> 27

ttgcctttgt agtatattcc tggtccacag cagagacggc aacttataga tgggtataat 60

<210> 28

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 28

tagattatcc caattatgta gtcatcactg agtcatcgtt ccaactgc 48

<210> 29

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 29

tcgactttaa acagcagaga cggcaactta taaagggtca cc 42

<210> 30

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 30

caatataagg aataaaaaga aacacggatg tagtcatcac tgagtcatcg cccaaagta 59

<210> 31

<211> 56

<212> DNA

<213> Artificial Sequence

<400> 31

agacctgcat gtgcttgatt gtgagcagca gagacggcaa cttatatccg gcccct 56

<210> 32

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 32

gggtggtatt gtaaaaatgc aggatgtagt catcactgag tcatcgcact gtttactac 59

<210> 33

<211> 56

<212> DNA

<213> Artificial Sequence

<400> 33

aagccaccaa agcaccgaga ggcagcagag acggcaactt atatagtgca accatg 56

<210> 34

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 34

gtggctcgaa tgtagtcatc actgagtcat cgtctgctca ct 42

<210> 35

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 35

ggctgaggca gcagagacgg caacttatag gagaatgg 38

<210> 36

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 36

atgtagtcat cactgagtca tcg 23

<210> 37

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 37

cagcagagac ggcaacttat a 21

<210> 38

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 38

tacggcgacc accgagatat atgttcacgc tcaccgtgcc cagtcgcg 48

<210> 39

<211> 63

<212> DNA

<213> Artificial Sequence

<400> 39

ggggatgttc tctattttgt attcttcatc tttcatatat ctcggtggtc gccgtaagaa 60

cat 63

<210> 40

<211> 57

<212> DNA

<213> Artificial Sequence

<400> 40

ggcgaccacc tacacctgaa catcaaatgc ttcatgaaag ctcacacatc ttccgcg 57

<210> 41

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 41

ggggaagagt tctattctgt atgcgtacac gctttgatgt tcaggtgtag gtggtcgcca 60

actctt 66

<210> 42

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 42

gcgaccacct agatcacact atgacatgtt ccaatcatcc atgccagcag acgcg 55

<210> 43

<211> 75

<212> DNA

<213> Artificial Sequence

<400> 43

ggggcggcct acgtgcgatc ggccgtccgg ctggccagct cttgtcatag tgtgatctag 60

gtggtcgcta ggccg 75

<210> 44

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 44

tgactacatg tctctatcga agtctttgac gtggtccgtg tgcacgcg 48

<210> 45

<211> 67

<212> DNA

<213> Artificial Sequence

<400> 45

ggggtcggcg cctccgtcac gctgcactgc acattctggc tcgatagaga catgtagtca 60

gcgccga 67

<210> 46

<211> 47

<212> DNA

<213> Artificial Sequence

<400> 46

agctgacaag gttcatgacg accattacca tgggtgatac cgccgcg 47

<210> 47

<211> 58

<212> DNA

<213> Artificial Sequence

<400> 47

ggggtattac ggagacggtg ctccgtgtgg tcgtcatgaa ccttgtcagc tcgtaata 58

<210> 48

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 48

gtcttgaagt cctgtgggat tcccaatgtt aatcagagtg tttgcaatga tcgcg 55

<210> 49

<211> 54

<212> DNA

<213> Artificial Sequence

<400> 49

gtcttgaagt cctgtgggat cattcaccag aagccagcat agatagagta cgcg 54

<210> 50

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 50

gtcttgaagt cctgtgggat gcatcatcag gcatagaaga tattgtactt gcgcg 55

<210> 51

<211> 58

<212> DNA

<213> Artificial Sequence

<400> 51

gcggagatag acataactga gaattccatc attctcttta ggtcaaaccc attgcgcg 58

<210> 52

<211> 77

<212> DNA

<213> Artificial Sequence

<400> 52

ggggctacgt ggtaagtgtt cagtcacgct cctttcgacc tgcgccgaat cccacaggac 60

ttcaagacaa cacgtag 77

<210> 53

<211> 52

<212> DNA

<213> Artificial Sequence

<400> 53

tgttagagtg cgagtcgtca atatagaatg cggctaacct taaccccgcg cg 52

<210> 54

<211> 68

<212> DNA

<213> Artificial Sequence

<400> 54

ggggggacgg atgtcttgat agttatagcc taattctata ttgacgactc gcactctaac 60

atccgtcc 68

<210> 55

<211> 56

<212> DNA

<213> Artificial Sequence

<400> 55

gccagtgaac tggcagaccg tcctgcgaca cagcagcagg aattaatgtt gccgcg 56

<210> 56

<211> 76

<212> DNA

<213> Artificial Sequence

<400> 56

ggggggccga gctccgcctg cgtcggagct acgccaactc ctttcaggac ggtctgccag 60

ttcactggcc tcggcc 76

<210> 57

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 57

ctacacgaca ctcttcctac aagctccgcc tcctgggttc acgcg 45

<210> 58

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 58

gggggtgaca tcgtgatact tctctactgt ctttttactg taggaagagt gtcgtgtaga 60

tgtcac 66

Claims

1. A multiplex nucleic acid detection system is characterized by comprising an amplification primer group and a detection probe group aiming at a Target nucleic acid sequence, wherein the detection probe group comprises a Target probe and a Beacon probe,

the Target probe comprises the following components from a 5 'end to a 3' end in sequence: 5 'end region, Target region and 3' end region;

the Beacon probe sequentially comprises the following components from a 5 'end to a 3' end: a 5 'end region, a loop region, a 3' end region;

LNA is modified at the 5 'end of the 5' end region sequence of the Target probe, C3 is modified at the 3 'end of the 3' end region sequence, and the Target region sequence can be reversely complementary with a Target nucleic acid sequence;

the 5 ' end of the sequence of the 5 ' end region of the Beacon probe is n continuous guanines, n is an integer of 1-8, the 3 ' end of the sequence of the 3 ' end region is modified with a fluorescent reporter group, and the loop region sequence is reversely complementary with the sequence of the 5 ' end region.

2. The multiplex nucleic acid detection system of claim 1, wherein the 3 'end of the sequence in the 3' end region of the Target probe is CGCG.

3. The multiple nucleic acid detection system of claim 1, wherein the length of the sequence in the 5 'end region of the Beacon probe is 5-8 bp, the length of the sequence in the 3' end region is 5-8 bp, and/or the length of the loop region is 30-50 bp.

4. The multiplex nucleic acid detecting system according to any one of claims 1 to 3, wherein the number n of guanine contiguous to the 5' end of the sequence in the 5 "terminal region of the Beacon probe is an integer of 3 to 5.

5. The multiplex nucleic acid detection system of any one of claims 1 to 4, wherein the amplification primer set comprises a CLO primer pair,

each CLO primer in the CLO primer pair sequentially comprises the following components from a 5 'end to a 3' end: a 5 'end region, a loop region, a 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.

6. The multiplex nucleic acid detection system of claim 5, wherein the amplification primer set further comprises a universal primer pair, wherein the upstream universal primer in the universal primer pair is identical to the loop region of the upstream CLO primer in the CLO primer pair; and 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.

7. The multiplex nucleic acid detecting system according to any one of claims 1 to 6, wherein the 3 '-end modified fluorescent reporter group of the sequence in the 3' -end region of the Beacon probe is selected from the group consisting of FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas, Red, LC RED640, Cy5, LC RED705, Alexa Fluor 488 and Alexa Fluor 750.

8. A multiplex nucleic acid detection method comprising the steps of:

obtaining nucleic acid of a biological sample to be detected;

9. The multiplex nucleic acid detection method of claim 8, wherein the reaction sequence of the PCR reaction is touchdown PCR.

10. The multiplex nucleic acid detection method according to any one of claims 8 to 9, wherein in the PCR reaction system, the concentration of each CLO primer is the same for different target nucleic acid sequences, the concentration of each universal primer is also the same, and the final concentration of each universal primer is 5 to 15 times, more preferably 10 times, the final concentration of each CLO single primer.

11. The multiplex nucleic acid detection method according to any one of claims 8 to 9, wherein in the PCR reaction system, the concentration of each Target probe for different Target nucleic acid sequences is the same, the concentration of each Beacon probe is the same, and the final concentration of each Target probe is 1 to 5 times, more preferably 2 times, the final concentration of the Beacon probe.

12. The multiplex nucleic acid detection method according to any one of claims 8 to 11, wherein the number of types of target nucleic acid sequences detectable by the method is 1 to 20.