Multiplex nucleic acid detection system and use thereof

Abstract

A multiplex nucleic acid detection system and a use thereof. The system comprises a Target probe and a common beacon probe. The Target probe sequentially comprises a probe primer region, a Tag exonuclease site, and a Target region, wherein the 5' end of the probe primer region is modified by a fluorescence quenching group 1; the 3' end of the probe primer region is connected to a fluorescent reporter group; and the 5' end of the Target region is modified by a fluorescent quenching group 2. The common beacon probe is divided into 10-20 primer binding regions by means of C3 Spacers. By utilizing the multiplex nucleic acid detection system, non-specific background peaks can be effectively eliminated, and false positive peaks possibly occurring in the presence of multi-channel targets are also eliminated, so that the false positive misjudgment rate of a result is reduced, thereby achieving more accurate, highly sensitive and highly specific detection of a target sequence.

Description

Multiplexed nucleic acid detection system and use thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of nucleic acid detection, and particularly relates to a multiplexed nucleic acid detection system and use thereof in preparing a respiratory disease diagnosis kit. BACKGROUND

[0002] There are various detection techniques in the field of nucleic acid detection, including probe molecule hybridization, nucleic acid-protein interaction, and sequencing technology. Among them, the polymerase chain reaction (PCR) based on the amplification principle of target sequences is the most widely used technical platform. Millions to billions of copies of specific DNA fragments are rapidly generated (amplified) and then further studied. The history of PCR dates back to the mid-1980s when the human genome project was being planned. It was a fundamental technology in the early stages of the human genome project. Today, PCR has evolved into a series of application methods and plays an important role, becoming an irreplaceable tool in many biological and medical researches.

[0003] In 1992, Higuchi proposed real-time fluorescent quantitative PCR technology. The progress of the entire PCR reaction was monitored in real time by detecting the content of ethidium bromide. After 1996, fluorescently labeled hydrolysis probes were officially reported to be applied to PCR. The fluorescence signal is collected after the extension of each PCR cycle, and the target gene in the sample to be tested is accurately quantified by using the mathematical function relationship of the PCR reaction (the number of cycles required to reach the exponential amplification period and the initial concentration of the template), the corresponding algorithm, and the use of standard samples.

[0004] This enhanced PCR method detects the amount of product formed during the reaction in real time by fluorescent dyes or fluorescence resonance energy transfer (FRET) probes. SYBR Green I is a fluorescent dye that only binds to DNA double strands. When it binds to DNA double strands, it emits fluorescence, and when it is released from the DNA molecule, the fluorescence decreases. Real-time PCR enables visualization of the initial amount of DNA in the reaction and the amount of DNA produced during the entire process. Compared with traditional culture methods, PCR-based detection methods have the advantages of fast speed, strong specificity, high sensitivity, etc.

[0005] The target sequence detection methods based on nucleic acid amplification mainly include:

[0006] 1. Using polyacrylamide gel electrophoresis, differentiating by fragment size, long manual operation time, and extremely low efficiency. 2. Molecular beacon method. The beacon molecule forms a hairpin structure in the liquid phase system, the distance between the fluorescent group and the quencher group is small, and FRET occurs; when the beacon hybridizes with the target sequence, the fluorescence and quencher group are separated and emit fluorescence. This method is limited to the matching of the molecular beacon and the target sequence at the same time, and the hairpin structure can be formed by itself. 3. Taqman probe method. The probe Tm value is set higher than the amplification primer, and the probe is first combined with the nucleic acid in the extension stage, and then the 5' exonuclease of the polymerase cuts the probe, releases the fluorescent group and the quencher group on the probe, and distinguishes the target sequence specific oligonucleotide probe by different fluorescent group markers. However, due to the type and number of equipment channels of the fluorescent markers, the number of multiple detection targets in a single tube reaction is limited. 4. Melting curve method. In real-time fluorescent quantitative PCR, a melting curve stage is added after the amplification cycle is completed, and the probe hybridizes with the target sequence to form a double strand. Based on this, the product can be identified or distinguished by fluorescence color and melting point. Target sequence, at the same time, with the increase of the detection target, the number of corresponding primers and probes increases, the fluorescence background increases, and the primer dimer formation is more complex.

[0007] Under the premise of meeting the detection needs of multiple targets, in order to improve the efficiency and control the economic and time cost, the multiplex PCR technology for simultaneously detecting multiple targets in a single tube reaction system has attracted widespread attention. In the traditional fluorescence beacon, non-specific background peaks and false positive peaks may occur in multiple detection, which affects interpretation and diagnosis. Therefore, it is necessary to develop a new detection system and method to eliminate the influence of non-specific background peaks and false positive peaks as much as possible.

[0008] SUMMARY

[0009] Based on this, the purpose of the present application is to provide a multiple nucleic acid detection system and its application. The multiple nucleic acid detection system of the present application is used for detection, which is more accurate and reliable.

[0010] The specific technical solutions for achieving the above-mentioned application purposes include the following.

[0011] The first aspect of the present application provides a multiple nucleic acid detection system, which comprises a probe combination, and the probe combination comprises a Target probe and a common beacon probe:

[0012] The Target probe comprises, from 5' end to 3' end, a probe primer region, a Tag exonuclease site and a Target region; the 5' end of the probe primer region is modified with a fluorescent quencher group 1; the 3' end of the probe primer region is connected with a fluorescent reporter group; the 5' end of the Target region is modified with a fluorescent quencher group 2; and the 3' end of the Target region comprises a nucleotide sequence which is reverse complementary to the target nucleic acid sequence.

[0013] The common beacon probe comprises, in sequence, a 5' end region, 10-20 primer binding regions and a 3' end region; the 10-20 primer binding regions are separated by C3 Spacers; the 3' end of each primer binding region has a sequence complementary to the 5' end of the probe primer region of the Target probe.

[0014] In a second aspect, the application provides use of the multiplex nucleic acid detection system in the preparation of a diagnostic kit for respiratory diseases.

[0015] The application has the following advantages:

[0016] In the multiplex nucleic acid detection system, the Target probe is designed as a double-fluorescence quenching structure, and the common beacon probe is designed with multiple 3' end C3 Spacers (each C3 Spacer is an extension block site), so that the common beacon probe is divided into 10-20 primer binding regions. With the combined action of these key technologies, the use amount of the common beacon probe is greatly reduced when the multiplex nucleic acid detection system is used for detection, the non-specific background peak does not appear in the melting curve analysis result, the non-specific false positive peak appearing in part of the channels when multiple channels of target sequences are detected can be effectively eliminated, and the target sequence can be accurately, sensitively and specifically detected. BRIEF DESCRIPTION OF DRAWINGS

[0017] Fig. 1 is a structural schematic diagram of the Target probe and the common beacon probe in the application.

[0018] Figs. 2-4 are working principle diagrams of the multiplex nucleic acid detection system of the application.

[0019] Fig. 5 is a partial melting curve diagram of detection of a negative sample by using a conventional Taqman probe and a conventional fluorescent probe (molecular beacon) in Comparative Example 1.

[0020] Fig. 6 is a partial melting curve diagram of detection of a negative sample by using the Target probe and the common beacon probe of the application in Comparative Example 1.

[0021] Fig. 7 is a melting curve diagram of detection of a qualified positive quality control sample by using the probe combination of the application and a conventional PCR system in Comparative Example 2.

[0022] Fig. 8 is a melting curve diagram of detection of a qualified positive quality control sample by using the probe combination of the application and the PCR system of the application in Comparative Example 2. DETAILED DESCRIPTION

[0023] For the purposes of the present invention, a more complete description will be provided below. 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 disclosure of the present invention is more thorough and complete.

[0024] Unless otherwise defined, 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 in this description, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0025] In the present invention, the inventors, based on in-depth research on gene detection technology, have developed a more efficient, convenient and accurate multiplex nucleic acid detection system. The detection system comprises a probe combination, amplification primers and a PCR system. The probe combination comprises a Target probe and a common beacon probe. The Target probe adopts a double-fluorescence quenching structure, which ensures that the fluorescence reporter group is always in a quenched state before the Target probe is not combined with a target sequence (the target sequence herein can be mutually interchangeable with the target nucleic acid sequence, which points to the same sequence), so that non-specific background peaks will not appear. By designing multiple 3' end C3 Spacers as extension blocking sites in the common beacon probe, the common beacon probe is divided into 10-20 primer binding regions (the length and sequence of each primer binding region are different, and the length is between 15-30 bases). Each common beacon probe can be recognized by multiple probe primers of different sequences (i.e. the probe primer region of the Target probe, the probe primer and the probe primer region can be mutually interchangeable herein, which points to the same sequence). Different sequence probe primer regions are combined with different primer binding regions and extended to produce extension products with different melting temperatures. In terms of input, in the traditional methodology, one fluorescent probe (i.e. molecular beacon) is consumed for one target (i.e. the length of the target sequence is between 20-200 bases). However, in the detection system of the present invention, one common beacon probe can combine 10-20 probe primer regions cut from the Target probe, greatly reducing the input of molecular beacon probes. In terms of detection results, each channel produces a single curve positive peak, and no non-specific background peaks and false positive peaks are detected. Therefore, the multiplex nucleic acid detection system of the present invention can accurately, highly sensitively and highly specifically detect target sequences.

[0026] In addition, in the multiple nucleic acid detection, the application also adds 0.01-0.2M (NH4) 2SO4 in the traditional PCR reaction system. The amplification by using the improved PCR reaction system can effectively eliminate the false positive peak of some channels in the detection of multiple channels of target sequences. The false positive peak may be caused by the poor specificity of Taqman enzyme in the traditional PCR reaction system, and the PCR is more prone to error, thereby generating a small amount of sequence that does not originally exist. If the sequence is recognized by the Target probe, a false positive peak will appear. The addition of ammonium sulfate can effectively improve the amplification specificity of Taqman enzyme, thereby reducing the error amplification rate in the PCR process and avoiding the appearance of false positive peaks.

[0027] Please refer to FIG. 2-4 (the probe primer in FIG. 2-4 points to the same sequence as the probe primer in this paper, and the probe beacon in FIG. 2-4 points to the same sequence as the probe beacon in this paper) for detailed understanding of the detection principle of the multiple nucleic acid detection system of the application: in the PCR amplification process of the target nucleic acid sequence (target sequence), if the probe primer region of the Target probe is not digested and separated from the Target region, the distance between the fluorescent quenching group 2 and the fluorescent reporter group is close, and the fluorescence of the fluorescent reporter group is quenched by the fluorescent quenching group 2; when the Target region sequence can be combined with the target sequence by complementary pairing, and when the amplified target nucleic acid sequence extends to the Tag enzyme exonuclease site of the Target probe, Taq enzyme will perform exonuclease function, so that the probe primer region is separated from the Target region and is in a free state (FIG. 2). The free probe primer region is in a curled state when it is not combined with the common beacon probe, and the terminal fluorescent reporter group and the fluorescent quenching group 1 are close to each other, and the fluorescence of the fluorescent reporter group is quenched by the fluorescent quenching group 1, and no fluorescent signal is generated; when the free probe primer region is combined with one of the primer binding regions of the common beacon probe by complementary pairing and begins to extend, the fluorescent reporter group at the 3' end of the probe primer region is separated from the fluorescent quenching group 1 (Quencher1) at the 5' end and generates a fluorescent signal (FIG. 3-4). The extension of the probe primer region by the common beacon probe will be prevented by the C3 Spacer, so as not to affect the complementary pairing of other primer binding regions on the common beacon probe and the probe primer region. After the melting curve analysis starts, the extension product of the probe primer region and the common beacon probe are separated at a specific temperature, so that the fluorescent signal decreases and a wave peak is generated.

[0028] As the target sequence PCR amplification to the plateau, the fluorescence signal accumulation to a certain value no longer increase, whereby the channel can be made of the target sequence amplification curve. Because the different probe primer region and the primer binding region of the common beacon probe length and sequence are different, the extension product of the complementary pairing is also different, which ensures that the different melting temperature of the extension product corresponds to the target sequence complementary to the target sequence, so that different melting peaks show the presence of corresponding target sequence. If the sequence of the target region of the target probe fails to bind to the target sequence (Off-Target), the probe primer region of the target probe cannot be excised (Figure 4). Because the 5' end of the target region of the fluorescence quencher 2 (Quencher2) and the 3' end of the fluorescence reporter group of the free probe primer region are always close to each other, the latter remains in a self-quenching state, so there is no background signal.

[0029] In some embodiments of the present application, a probe combination for detecting multiple nucleic acids is disclosed, which comprises a target probe (Primer Probe) and a common beacon probe (Common Beacon);

[0030] (1) The target probe (structure diagram as shown in Figure 1) comprises, from 5' end to 3' end, probe primer region, Taq exonuclease site and target region; the 5' end of the probe primer region is modified with fluorescence quencher 1 (Quencher1) to provide fluorescence quenching function; the probe primer region comprises nucleotide sequence which is not complementary to the target nucleic acid sequence; the 3' end is connected with fluorescence reporter group (Reporter Dye); the 5' end of the target region is modified with fluorescence quencher 2 (Quencher2) to provide fluorescence quenching function; the 3' end of the target region comprises nucleotide sequence which is reverse complementary to the target nucleic acid sequence, so that the target probe can bind to the target sequence by complementary pairing; the 5' end of the probe primer region and the 3' end of the target region are non-homologous sequences and do not bind to the template;

[0031] (2) The common beacon probe (structure diagram as shown in Figure 1) provides a fluorescence signal release system to realize melting curve analysis, which comprises, in order:

[0032] 5' end region (composed of 5-8 bases)

[0033] 10-20 Binding Regions: Each of the Binding Regions (length of 15-35 bases, different sequences) is separated by a C3 Spacer, each of which serves as an extension block site, and each of the 3' ends of the Binding Regions has a sequence complementary to a different probe primer region of the Target probe;

[0034] 3' end region (5-8 bases), the end of which is 4 consecutive G bases, for protecting the DNA structure stability of the common beacon probe.

[0035] In some embodiments, each of the fluorescent reporter groups 1 and 2 is selected from the group consisting of FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas Red, LC RED 640, Cy5, LC RED 705, Alexa Fluor 488, and Alexa Fluor 750.

[0036] In some embodiments, the fluorescent quencher group 1 and the fluorescent quencher group 2 can be the same group or different groups.

[0037] In some embodiments, the system for detecting multiple nucleic acids further comprises amplification primers for amplifying the target nucleic acid sequences, including an upstream primer and a downstream primer, each of which has a 5' end sequence length of 18-25 bp and a higher Tm value, and each of which has a 3' end sequence length of 10-15 bp and a lower Tm value. Each of the upstream primer and the downstream primer comprises a nucleotide sequence complementary to the target nucleic acid sequence.

[0038] In some embodiments, the probe set comprises Target probes and common beacon probes for detecting 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, at least 20, or at least 20 or more target nucleic acid sequences.

[0039] In some embodiments, the amplification primers comprise amplification primers for amplifying 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, at least 20, or at least 20 or more target nucleic acid sequences.

[0040] In some embodiments of the present application, the multiplex nucleic acid detection system is used in the preparation of a respiratory disease detection kit.

[0041] In some embodiments of the present application, the multiplex nucleic acid detection system is used in the preparation of a respiratory disease detection kit.

[0042] In some embodiments of the present application, the kit further comprises (NH4)2SO4.

[0043] In some embodiments of the present application, a method for detecting multiplex nucleic acid is disclosed, comprising the following steps:

[0044] (1) designing the multiplex nucleic acid detection system according to the pathogen to be detected;

[0045] (2) mixing the nucleic acid of the biological sample to be detected, DNA polymerase and the multiplex nucleic acid detection system of step (1) to prepare a PCR reaction system, and then performing PCR reaction and melt curve analysis.

[0046] In some embodiments of the present application, the working concentration of the amplification primer in the PCR reaction system is 0.3 pmol / μL-2.0 pmol / μL, the working concentration of the Target probe is 1.0 pmol / μL-3.0 pmol / μL, the working concentration of the common beacon probe is 0.2 pmol / μL-2.2 pmol / μL, and the working concentration of (NH4)2SO4 is 0.01-0.2 M. The addition of ammonium sulfate in the reaction system can effectively eliminate the false positive peaks in some channels when detecting multiple channels of target sequences.

[0047] In some embodiments of the present application, the pathogen is a pathogen of respiratory infection, digestive tract infection, blood infection and / or urinary tract infection. Further preferably, the pathogen is a pathogen of respiratory infection with similar symptoms such as cough, which can be influenza virus such as influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, Mycoplasma pneumoniae, parainfluenza virus, etc.

[0048] In some embodiments of the present application, the biological sample to be detected can be selected from, but not limited to, serum sample, plasma sample, whole blood sample, sputum sample, swab sample, lavage fluid sample, fresh tissue sample, formalin-fixed paraffin-embedded tissue (FFPE) sample, urine sample, bacterial culture, virus culture, cell line culture, and artificially synthesized plasmid sample.

[0049] In some embodiments of the present application, the nucleic acid of the biological sample to be detected is deoxyribonucleic acid or ribonucleic acid. When the biological sample is ribonucleic acid, the reaction system further comprises reverse transcriptase, and the reaction procedure further comprises reverse transcription PCR.

[0050] The present application will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0051] Example 1 Multiple nucleic acid detection system and detection method

[0052] The present embodiment provides a method for detecting pathogens of a biological sample to be tested using a multiple nucleic acid detection system, comprising the following steps:

[0053] I. Designing a multiple nucleic acid detection system

[0054] According to the literature review and research, the nucleic acid sequence conserved segment of the pathogen to be detected (including new crown ROF1ab, new crown N gene, influenza A virus, influenza B virus, Bordetella pertussis, Bordetella parapertussis, respiratory syncytial virus, Chlamydia pneumoniae, rhinovirus, adenovirus, metapneumovirus, bocavirus, coronavirus, parainfluenza virus I, parainfluenza virus II and HBB internal reference gene) is determined, at least one specific target gene sequence (target nucleic acid sequence) is selected, and based on the selected specific target gene sequence, a multiple nucleic acid detection system (including amplification primer, Target probe and common beacon probe) is designed. The sequences of the upstream primer F n , the downstream primer R n , the Target region T n in the Target probe, the sequence P n of the probe primer region in the Target probe and the sequence S n of the common beacon probe are used. Specifically as shown in Table 1.

[0055] Table 1

[0056] II. Obtain the nucleic acid of the biological sample to be tested;

[0057] III. Mix the nucleic acid of the biological sample to be tested, DNA polymerase and the multiple nucleic acid detection system of Table 1 to prepare a PCR reaction system and perform PCR reaction.

[0058] The PCR reaction system is shown in Table 2 and the PCR reaction program is shown in Table 3.

[0059] Table 2

[0060] Table 3

[0061] IV. Melting curve analysis

[0062] The types of target nucleic acid sequences present in the reaction system are determined by melt curve analysis of the PCR product, thereby achieving multiplex nucleic acid detection. The Tm value ranges of different pathogens in each channel are shown in Table 4.

[0063] Table 4

[0064] Influence of the traditional Taqman probe and the Target probe of the present application on the detection results

[0065] In the present comparative example, a negative sample (sterile nucleic acid-free water extracted nucleic acid) (i.e. a biological sample to be tested) is detected by using a multiplex nucleic acid detection system comprising a traditional Taqman probe and a multiplex nucleic acid detection system comprising a probe combination (Target probe and common beacon probe) of the present application. The PCR reaction system is prepared according to Table 2, and the PCR reaction is carried out according to the process of Table 3. The PCR reaction product is obtained, and then melt curve analysis is carried out on the PCR product. The target pathogens are detected in the FAM, VIC, ROX, Cy5, and Cy5.5 channels. When there is a melting peak in the Tm reference value range of a specific pathogen and the peak height is more than 8, then according to Table 4, the pathogen in the Tm range is determined to be positive. When two or more melting peaks appear at the same time, it is determined that the biological sample to be tested is infected with two or more pathogens at the same time, and then the types of target nucleic acid sequences present in the biological sample to be tested are determined.

[0066] The traditional Taqman probe comprises a probe primer region and a Target region, and does not comprise a probe beacon region, nor does it carry a fluorescent group (a fluorescent quencher group and a fluorescent reporter group). The sequence of the Target region is the same as that of the Target region of the Target probe of the present application. When the multiplex nucleic acid detection system comprising the traditional Taqman probe is used for detection, a fluorescent probe (equivalent to the common beacon probe of the present application) for melt curve analysis needs to be additionally added in the reaction system of Table 2. The concentration of the Taqman probe and the common beacon probe is 20 nM, the volume ratio is 2:1, and the total volume is 1 μL. The 5' end of the fluorescent probe comprises a fluorescent reporter group, and the 3' end comprises a fluorescent quencher group. Before the fluorescent probe is combined by complementary pairing with the probe primer region of the Taqman probe, the fluorescent probe is in a curled state, and the fluorescent reporter group is quenched by the close fluorescent quencher group and does not emit a fluorescent signal. When the probe primer region is combined with the target sequence and starts to extend, the configuration of the fluorescent probe changes, the 5' end and the 3' end are separated, and the fluorescent reporter group starts to emit a signal.

[0067] Figure 5 shows part of the results of detecting a negative sample using the conventional Taqman probe before improvement. Since the biological sample to be detected is a negative sample with nucleic acid extracted from sterile nucleic acid-free water, there should be no melting peak in the melting curve. However, as shown in Figure 5, low melting peaks appear in the same Tm range in each channel, which actually result from the background fluorescence signal in the system, and are non-specific background peaks. Since the peak height of the background peak in some channels (such as FAM) is higher than the positive determination threshold (8.0), the background peak will cause false positive misjudgment of the results in the channel, which will interfere with the accuracy of the final diagnosis results.

[0068] When the negative sample is detected using the multiplex nucleic acid detection system of Example 1 of the present application, the results are shown in Figure 6. As can be seen from Figure 6, no melting peaks as shown in Figure 5 appear in the melting curve of each channel. It can be seen that the multiplex nucleic acid detection system of the present application does not produce non-specific background peaks, so it will not cause misleading judgment of the results, and the results are more accurate and reliable.

[0069] Effect of the improved multiplex nucleic acid detection system of Comparative Example 2 on the detection results in combination with the conventional PCR system

[0070] This comparative example uses the multiplex nucleic acid detection system of Example 1 of the present application in combination with the conventional PCR system without adding ammonium sulfate to detect positive standard samples of influenza B virus (FAM 83-87°C), Bordetella parapertussis (VIC 74-78°C), human metapneumovirus (ROX 79-83°C), and coronavirus (Cy568-72°C), and analyzes the melting curves thereof. The target sequences of the four pathogens are selected from the FAM, VIC, ROX, and Cy5 channels, and the Tm values of the melting curves of the target sequences do not coincide.

[0071] The nucleic acids of the positive standard samples are extracted, and the PCR reaction systems are prepared according to Table 5 (conventional PCR system) and Table 2, respectively. The PCR reaction is performed according to the process of Table 3, and the PCR reaction products are obtained.

[0072] Table 5

[0073] The PCR products are subjected to melting curve analysis of each channel, so as to determine the types of target nucleic acid sequences present in the reaction system. The target pathogens are detected by the FAM, VIC, ROX, and Cy5 channels, and when there is a melting peak in the Tm reference value range of a specific pathogen and the peak height is higher than 8, then according to Table 4, the pathogen in the Tm range is determined to be positive; when two or more melting peaks appear at the same time, then it is determined that the sample is infected with two or more pathogens at the same time.

[0074] When the PCR reaction system of Table 5 was used to detect the positive standard nucleic acid, the results are shown in Figure 7. As shown in Figure 7, a total of 5 melting peaks belonging to 4 channels were observed, including Cy5 71-72℃, ROX 81-82℃, FAM 86-87℃, VIC 76-77℃, and FAM 76-77℃, corresponding to the pathogens: coronavirus, human metapneumovirus, influenza B virus, Bordetella parapertussis, and novel coronavirus (N gene). However, the selected positive standard sample did not contain novel coronavirus (N gene), and the peak value of the corresponding FAM 76-77℃ positive peak was low, so it was suspected that the FAM 76-77℃ channel positive peak was a false positive peak.

[0075] When the PCR reaction system of Table 2 was used to detect the positive standard nucleic acid, the results are shown in Figure 8. As shown in Figure 8, a total of 4 melting peaks belonging to 4 channels were observed, including Cy5 71-72℃, ROX 81-82℃, FAM 86-87℃, and VIC 76-77℃, corresponding to the pathogens: coronavirus, human metapneumovirus, influenza B virus, and Bordetella parapertussis, which is completely consistent with the positive target sequence input, and the FAM 76-77℃ positive peak shown in Figure 7 does not appear. Therefore, the FAM 76-77℃ channel positive peak in Figure 7 is a false positive peak, and by improving the PCR reaction system, the false positive peak no longer appears.

[0076] Therefore, using the PCR reaction system of the present application for multiplex nucleic acid detection can eliminate the problem of false positive interpretation caused by background peaks, while ensuring that false positive peaks no longer appear in the presence of multiple channel target sequences, making the interpretation of positive results more accurate.

[0077] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not contradict, they should be considered within the scope of the present disclosure.

[0078] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but should not be construed as limiting the scope of the patent. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present patent should be subject to the appended claims.

Claims

1. A multiplexed nucleic acid detection system, comprising: The system comprises a probe combination comprising a Target probe and a common beacon probe; The Target probe comprises, from 5' end to 3' end, a probe primer region, an exonuclease site of Tag enzyme and a Target region; the 5' end of the probe primer region is modified with a fluorescence quenching group 1; the 3' end of the probe primer region is connected with a fluorescence reporter group; the 5' end of the Target region is modified with a fluorescence quenching group 2; and the 3' end of the Target region comprises a nucleotide sequence reverse complementary to the target nucleic acid sequence; The common beacon probe comprises, from 5' end to 3' end, a 5' end region, 10-20 primer binding regions and a 3' end region; each primer binding region is separated by a C3 Spacer; and the 3' end of each primer binding region has a sequence complementary to the 5' end of the probe primer region of the Target probe.

2. The multiplexed nucleic acid detection system of claim 1, wherein, The sequence of the Target region of the Target probe is reverse complementary to the target nucleic acid sequence.

3. The multiplexed nucleic acid detection system of claim 1, wherein, The 3' end region of the common beacon probe consists of 5-8 bases, and the terminal end is four continuous G bases.

4. The multiplexed nucleic acid detection system of claim 1, wherein, The length and sequence of each primer binding region of the common beacon probe are different; and / or, the length of each primer binding region is 15-35 bases.

5. The multiplexed nucleic acid detection system of claim 1, wherein, The fluorescence reporter group, the fluorescence quenching group 1 and the fluorescence quenching group 2 are selected from FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas, Red, LC RED640, Cy5, LC RED705, Alexa Fluor 488 and Alexa Fluor 750; and / or, the fluorescence quenching group 1 and the fluorescence quenching group 2 are the same or different.

6. The multiplexed nucleic acid detection system according to any one of claims 1 to 5, wherein The detection system further comprises amplification primers for amplifying the target nucleic acid sequence; and / or, the sequence length of the 5' end of the amplification primers is 18-25 bp; and the sequence length of the 3' end of the amplification primers is 10-15 bp.

7. A diagnostic kit for respiratory diseases, characterized in that, The detection system comprises the multiplexed nucleic acid according to any one of claims 1-6.

8. The respiratory disease detection kit according to claim 7, characterized in that, It further comprises (NH4)2SO4.

9. A method of detecting multiple nucleic acids, characterized by, The detection kit according to claim 8 is used for performing PCR reaction on the sample to be tested, and then performing melting curve analysis.

10. The method of claim 9, wherein the detecting is performed by a method selected from the group consisting of gel electrophoresis, capillary electrophoresis, microarray, mass spectrometry, and a combination thereof. In the reaction system of the PCR reaction, the working concentration of the Target probe is 1.0 pmol / μL-3.0 pmol / μL, the working concentration of the amplification primers is 0.3 pmol / μL-2.0 pmol / μL, the working concentration of the common beacon probe is 0.2 pmol / μL-2.2 pmol / μL, and the working concentration of (NH4)2SO4 is 0.01-0.2 M.

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