Multiple nucleic acid detection system and its application

Abstract

The present invention discloses a multiple nucleic acid detection system and its application, the system includes a Target probe and a public beacon probe; the Target probe includes: a probe primer region, a Tag enzyme exonuclease site and a Target region in sequence; the 5' end of the probe primer region is modified with a fluorescent quenching 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 quenching group 2; the public beacon probe is separated into 10 to 20 primer binding regions by C3Spacer. The multiple nucleic acid detection system of the present invention can effectively eliminate non-specific background peaks, and at the same time eliminate false positive peaks that may appear in the presence of multi-channel targets, thereby reducing the false positive misjudgment rate of the results, and realizing more accurate, highly sensitive, and highly specific detection of target sequences.

Description

Technical Field

[0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a multiple nucleic acid detection system and an application thereof in preparing a respiratory disease diagnosis kit. Background Art

[0002] In the field of nucleic acid detection, there are many detection technology routes including probe molecule hybridization, nucleic acid-protein interaction and sequencing technology. Among them, the polymerase chain reaction (PCR) based on the principle of target sequence amplification is the most widely used technology platform. By rapidly generating (amplifying) millions to billions of copies of specific DNA fragments, and then further studying them. The history of PCR can be traced back to the mid-1980s when the Human Genome Project was brewing. It was a basic technology in the early days 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 research.

[0003] In 1992, Higuchi proposed real-time fluorescence quantitative PCR technology. The progress of the entire PCR reaction is 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. After the extension of each PCR cycle, the fluorescence signal is collected, and the target gene in the sample to be tested is accurately quantified through the mathematical function relationship of the PCR reaction (the number of cycles required to reach the exponential amplification phase and the initial concentration of the template), the corresponding algorithm, and the use of standard products.

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

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

[0006] 1. Using polyacrylamide gel electrophoresis to distinguish fragments based on their size, manual operation takes a long time and is extremely inefficient.

[0007] 2. Molecular beacon method. The beacon molecule forms a hairpin structure in the liquid phase system. The distance between the fluorescent group and the quenching group is small, and FRET occurs. When the beacon hybridizes with the target sequence, the fluorescent and quenching groups separate and emit fluorescence. This method is limited by the fact that the molecular beacon can form a hairpin structure while matching the target sequence. 3. Taqman probe method. The probe Tm value is set higher than the amplification primer. In the extension stage, the probe first binds to the nucleic acid, and then the polymerase 5' exonuclease cuts the probe to release the fluorescent group and quenching group on the probe. Different fluorescent group labels are used to distinguish the oligonucleotide probes that are specific to the target sequence. However, due to the type of fluorescent labeling and the number of equipment channels, the number of targets for multiple detection in a single tube reaction is limited. 4. Melting curve method. In real-time fluorescence 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 product, the target sequence can be identified or distinguished by fluorescent color and melting point. At the same time, as the number of detection targets increases, the number of corresponding primers and probes increases, the fluorescence background increases, and the formation of primer dimers becomes more complicated.

[0008] Under the premise of meeting the detection needs of multiple targets, in order to improve efficiency and control economic and time costs, multiplex PCR technology for simultaneous detection of multiple targets in a single-tube reaction system has attracted widespread attention. Traditional fluorescent beacons will produce non-specific background peaks and false positive peaks in multiple detections, affecting interpretation and diagnosis. Therefore, it is necessary to develop new detection systems and methods to eliminate the influence of non-specific background peaks and false positive peaks as much as possible. Summary of the invention

[0009] Based on this, the object of the present invention is to provide a multiple nucleic acid detection system and its application, using

[0010] The multiple nucleic acid detection system of the present invention is more accurate and reliable for detection.

[0011] The specific technical solutions for achieving the above-mentioned invention objectives include the following.

[0012] In a first aspect of the present invention, a multiple nucleic acid detection system is provided, the system comprising a probe combination, the probe combination comprising a target probe and a common beacon probe:

[0013] The Target probe includes, from the 5' end to the 3' end, a probe primer region, a Tag enzyme exonuclease site and a Target region; the 5' end of the probe primer region is modified with a fluorescent quenching 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 quenching group 2; the 3' end of the Target region contains a nucleotide sequence that is reverse complementary to the target nucleic acid sequence;

[0014] The public beacon probe includes: a 5' end region, 10 to 20 primer binding regions and a 3' end region in sequence; the 10 to 20 primer binding regions are separated by C3Spacer; the 3' end of each primer binding region has a sequence that can complement the 5' end of the probe primer region of the primer Target probe.

[0015] The second aspect of the present invention provides the use of the above-mentioned multiple nucleic acid detection system in the preparation of a respiratory disease diagnosis kit.

[0016] The present invention has the following beneficial effects:

[0017] The multiple nucleic acid detection system of the present invention designs the Target probe as a dual fluorescence quenching structure and designs multiple 3'-end C3Spacers (each C3Spacer is an extension blocking site) in the public beacon probe, thereby dividing the public beacon probe into 10 to 20 primer binding regions. Under the joint action of these key technologies, when the multiple nucleic acid detection system of the present invention is used for detection, the amount of the public beacon probe is greatly reduced, and the melting curve analysis result will not have non-specific background peaks, and the non-specific false positive peaks that appear in some channels when detecting multi-channel target sequences can be effectively eliminated, and the target sequence can be accurately, highly sensitively and highly specifically detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the structure of the Target probe and the public beacon probe in the present invention.

[0019] Figures 2 to 4 Schematic diagram of the working principle of the multiple nucleic acid detection system of the present invention.

[0020] Figure 5 This is a partial melting curve diagram of the negative sample detected using the traditional Taqman probe and the traditional fluorescent probe (molecular beacon) in Comparative Example 1.

[0021] Figure 6 This is a partial melting curve diagram of the negative sample detected by using the Target probe and the public beacon probe of the present invention in Comparative Example 1.

[0022] Figure 7 This is a melting curve diagram of comparative example 2 in which the probe combination of the present invention is used in combination with a traditional PCR system to detect qualified positive quality control products.

[0023] Figure 8 This is a melting curve diagram of the qualified positive quality control product detected by using the probe combination of the present invention in combination with the PCR system of the present invention in Comparative Example 2. DETAILED DESCRIPTION

[0024] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0025] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0026] In the present invention, based on the in-depth study of gene detection technology, the inventors have developed a more efficient, convenient and accurate multiple nucleic acid detection system, which includes a probe combination, an amplification primer and a PCR system, wherein the probe combination includes a Target probe and a public beacon probe, wherein the Target probe adopts a dual fluorescence quenching structure to ensure that the fluorescent reporter group is always in a quenching state before the Target probe is bound to the target sequence (the target sequence and the target nucleic acid sequence in this article can be interchangeable with each other and point to the same sequence), so that no non-specific background peak will appear; by designing multiple 3'-end C3Spacers as extension blocking sites in the public beacon probe, the public beacon probe is divided into 10 to 20 primer binding regions (the length and sequence of each primer binding region are different, and the length is between 15 and 30 bases), each public 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 in this article can be interchangeable with each other and point to the same sequence), and the probe primer regions of different sequences are thus combined with different primer binding regions and extended to produce extension products with different melting temperatures. From the perspective of input, in traditional methodology, a primer probe that hits the target (i.e., the length of the target sequence is between 20 and 200 bases) needs to consume a fluorescent probe (i.e., a molecular beacon), but in the detection system of the present invention, a public beacon probe can bind to 10 to 20 probe primer regions cut from the target probe, greatly reducing the input of the molecular beacon probe; from the detection results, each channel produces a single curve positive peak, and no nonspecific background peaks and false positive peaks are detected. Therefore, the multiple nucleic acid detection system of the present invention can accurately, highly sensitively, and highly specifically detect the target sequence.

[0027] In addition, when performing multiple nucleic acid detection, the present invention also adds 0.01-0.2M (NH4)2SO4 to the traditional PCR reaction system. The use of an improved PCR reaction system for amplification can effectively eliminate the false positive peaks that appear in some channels when detecting multi-channel target sequences. This may be because the amplification specificity of the Taqman enzyme under the traditional PCR reaction system is poor, and PCR is more prone to errors, thereby generating a small amount of sequences that did 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 the Taqman enzyme, thereby reducing the error amplification rate during the PCR process and avoiding the appearance of false positive peaks.

[0028] Please refer to Figure 2 to Figure 4 ( Figure 2 to Figure 4 The probe primer in the present invention points to the same sequence as the probe primer region in the present invention. Figure 2 to Figure 4 The probe beacon in the Target probe points to the same sequence as the probe beacon region in this article), the detection principle of the multiple nucleic acid detection system of the present invention can be understood in detail: during the PCR amplification of the target nucleic acid sequence (target sequence), if the probe primer region of the Target probe is not cleaved and separated from the Target region, the fluorescence quenching group 2 is close to the fluorescence reporter group, and the fluorescence of the fluorescence reporter group is quenched by the fluorescence quenching group 2; when the Target region sequence can bind to the target sequence through complementary pairing, and when the amplified target nucleic acid sequence extends to the Tag enzyme exoclease site of the Target probe, the Taq enzyme will perform the exoclease function, so that the probe primer region is separated from the Target region and is in a free state ( Figure 2 ). When the public beacon probe is not bound, the free probe primer region is in a curled state, the fluorescent reporter group at the end and the fluorescent quencher group 1 are close to each other, the fluorescence of the fluorescent reporter group is quenched by the fluorescent quencher group 1, and no fluorescent signal is generated; when the free probe primer region binds to one of the primer binding regions of the public beacon probe through complementary pairing and begins to extend, the fluorescent reporter group at the 3' end of the probe primer region and the fluorescent quencher group 1 (Quencher1) at the 5' end separate and generate a fluorescent signal ( Figure 3-4 ). The extension of the probe primer region in the public beacon probe will be blocked by C3Spacer, so as not to affect the complementary pairing of other primer binding regions on the public beacon probe and the probe primer region. After the melting curve analysis begins, the extension product of the probe primer region and the public beacon probe unwind the double strand at a specific temperature, thereby causing the fluorescence signal to decrease and generate a peak.

[0029] As the target sequence PCR amplification reaches the plateau phase, the fluorescent signal accumulates to a certain value and then stops increasing, thus making an amplification curve (AmplificationCurve) of the target sequence in this channel. Since the lengths and sequences of different probe primer regions and the primer binding regions of the common beacon probe are different, the melting curves of the extension products after complementary pairing of the two are also different, ensuring that the different melting temperatures of the extension products correspond one-to-one to the target sequences of the complementary pairing of the Target region, so that different melting peaks show the presence of the corresponding target sequences. If the Target region sequence of the Target probe fails to bind to the target sequence through complementary pairing (Off-Target), the probe primer region of the Target probe cannot be exonucleated ( Figure 4 ). Since the fluorescent quencher 2 (Quencher2) at the 5' end of the Target region is always close to the fluorescent reporter group at the 3' end of the undissociated probe primer region, the latter remains in a self-quenching state, so no background signal will appear.

[0030] In some embodiments of the present invention, a probe combination for detecting multiple nucleic acids is disclosed, the probe combination comprising a Target probe (PrimerProbe) and a common beacon probe (CommonBeacon);

[0031] (1) Target probe (structural diagram as shown in Figure 1 The 5' end of the probe primer region is modified with a fluorescent quenching group 1 (Quencher1) to provide a fluorescent quenching function; the probe primer region contains a nucleotide sequence that is non-hybridized and complementary to the target nucleic acid sequence; the 3' end is connected with a fluorescent reporter group (ReporterDye); the 5' end of the Target region is modified with a fluorescent quenching group 2 (Quencher2) to provide a fluorescent quenching function; the 3' end of the Target region contains a nucleotide sequence that is reversely complementary to the target nucleic acid sequence, so that the Target probe can bind to the target sequence through complementary pairing; the 5' end of the probe primer region and the 3' end of the Target region are both non-homologous sequences and do not bind to the template;

[0032] (2) CommonBeacon (structural diagram as shown in Figure 1 As shown), a fluorescent signal release system is provided to realize melting curve analysis, and the public beacon probe comprises:

[0033] 5' terminal region (consisting of 5 to 8 bases)

[0034] 10 to 20 primer binding regions (BindingRegion): Each of the primer binding regions (15 to 35 bases in length, with different sequences) is separated by C3Spacer, each C3Spacer serves as an extension blocking site, and the 3' end of each primer binding region has a sequence that can complement a different probe primer region of the Target probe;

[0035] The 3' terminal region (5 to 8 bases) has four consecutive G bases at the end, which are used to protect the DNA structure stability of the public beacon probe.

[0036] In some of the embodiments, the fluorescent reporter groups 1 and 2 are selected from FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas, Red, LCRED640, Cy5, LCRED705, AlexaFluor488 and AlexaFluor750.

[0037] In some embodiments, the fluorescence quenching group 1 and the fluorescence quenching group 2 may be the same group or different groups.

[0038] In some embodiments, the system for detecting multiple nucleic acids further includes amplification primers for amplifying target nucleic acid sequences, including upstream primers and downstream primers, wherein the sequence lengths of the 5′ ends of the upstream primers and downstream primers are both 18 to 25 bp, with a higher Tm value; the sequence lengths of the 3′ ends of the upstream primers and downstream primers are both 10 to 15 bp, with a lower Tm value. The upstream primers and downstream primers both contain nucleotide sequences that are complementary to the target nucleic acid sequences.

[0039] In some embodiments, the probe combination includes 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 more target nucleic acid sequences.

[0040] In some embodiments, the amplification primers include 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 more target nucleic acid sequences.

[0041] In other embodiments of the present invention, the use of the above-mentioned multiple nucleic acid detection system in the preparation of a respiratory disease detection kit is disclosed.

[0042] In other embodiments of the present invention, a respiratory disease detection kit is disclosed, comprising the above-mentioned multiple nucleic acid detection system.

[0043] In some embodiments, the kit further comprises (NH4)2SO4.

[0044] In other embodiments of the present invention, a method for detecting multiple nucleic acids is disclosed, comprising the following steps:

[0045] (1) Designing the above-mentioned multiple nucleic acid detection system for the pathogen to be detected;

[0046] (2) Mix the nucleic acid of the biological sample to be tested, DNA polymerase and the multiple nucleic acid detection system described in step (1) to prepare a PCR reaction system, perform a PCR reaction, and then perform a melting curve analysis.

[0047] In some embodiments, in the PCR reaction system, the working concentration of the amplification primer is 0.3 pmol / μL to 2.0 pmol / μL, the working concentration of the target probe is 1.0 pmol / μL to 3.0 pmol / μL; the working concentration of the public beacon probe is 0.2 pmol / μL to 2.2 pmol / μL, and the working concentration of (NH4)2SO4 is 0.01 to 0.2 M. Adding ammonium sulfate to the reaction system can effectively eliminate the false positive peaks that appear in some channels when detecting multi-channel target sequences.

[0048] In some embodiments, the pathogen is a pathogen of respiratory tract infection, digestive tract infection, blood infection and / or urinary tract infection. More preferably, the pathogen is a pathogen of respiratory tract infection with similar symptoms such as cough, and can be an influenza virus such as influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, Mycoplasma pneumoniae, parainfluenza virus, etc.

[0049] In some embodiments, the biological sample to be tested can 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, and artificially synthesized plasmid samples.

[0050] In some embodiments, the nucleic acid of the biological sample to be tested 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.

[0051] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0052] Example 1 Multiple Nucleic Acid Detection System and Detection Method

[0053] This embodiment provides a method for detecting pathogens in a biological sample using a multiple nucleic acid detection system, comprising the following steps:

[0054] 1. Design of multiple nucleic acid detection system

[0055] According to the literature review and research, the conserved nucleic acid sequence segments of the pathogens to be detected (including ROF1ab of the new coronavirus, N gene of the new coronavirus, 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) are determined, and 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 primers, target probes and public beacon probes) is designed. The upstream primer F used n , downstream primer R n Target probe in the Target region T n The sequence of the probe primer region in the Target probe is P n and the sequence S of the public beacon probe n The details are shown in Table 1.

[0056] Table 1

[0057]

[0058]

[0059]

[0060]

[0061] 2. Obtaining nucleic acid from biological samples to be tested;

[0062] 3. Mix the nucleic acid of the biological sample to be tested, DNA polymerase and the multiple nucleic acid detection system in Table 1 to prepare a PCR reaction system, and perform a PCR reaction.

[0063] The PCR reaction system is shown in Table 2, and the PCR reaction program is shown in Table 3.

[0064] Table 2

[0065] Components Add volume 5×PCRBuffer 5μL DNA polymerase (5U / μL) 5μL dNTPs(20mM) 0.5μL Reverse transcriptase (200 U / μL) 0.5μL UDG enzyme (1U / μL) 0.5μL RNaseInhibitor (40U / μL) 0.5μL Amplification primer mixture (0.8 pmol / μL each) 1μL Target probe (1.6 pmol / μL each) 1μL Public beacon probe (1.0pmo / μL) 1μL <![CDATA[MgCl2]]> 5mM KCl 0.125M <![CDATA[(NH4)2SO4]]> 0.02M Purified water Add water to the reaction system to 20 μL

[0066] Table 3

[0067]

[0068]

[0069] 4. Melting Curve Analysis

[0070] By performing melting curve analysis on the products obtained from the above PCR reaction, the types of target nucleic acid sequences present in the reaction system can be determined to achieve multiple nucleic acid detection. The Tm value ranges of different pathogens in each channel are shown in Table 4.

[0071] Table 4

[0072]

[0073] Comparative Example 1 Effects of the Traditional Taqman Probe and the Target Probe of the Present Invention on the Detection Results

[0074] In this comparative example, a multiple nucleic acid detection system including a traditional Taqman probe and a multiple nucleic acid detection system including the probe combination of the present invention (Target probe and public beacon probe) are used to detect negative samples (nucleic acid extracted from sterile nucleic acid-free water) (i.e., the biological sample to be tested), the PCR reaction system is prepared according to Table 2, and the PCR reaction is carried out according to the process of Table 3 to obtain a PCR reaction product, and then the above-mentioned PCR product is subjected to melting curve analysis, and the target pathogen is detected using FAM, VIC, ROX, Cy5, and Cy5.5 channels. When there is a melting peak within the Tm reference value range of a specific pathogen and the peak height exceeds 8, the pathogen within the Tm range is determined to be positive according to Table 4; when two or more melting peaks appear at the same time, it is determined that the biological sample to be tested is simultaneously infected with two or more pathogens, and then the type of target nucleic acid sequence present in the biological sample to be tested is determined.

[0075] The traditional Taqman probe includes a probe primer region and a Target region, but does not include a probe beacon region, nor does it carry a fluorescent group (a fluorescent quenching group and a fluorescent reporter group); wherein the sequence of the Target region is the same as the sequence of the Target region of the Target probe of the present invention. When using a multiple nucleic acid detection system comprising a traditional Taqman probe for detection, a fluorescent probe for melting curve analysis (equivalent to the public beacon probe of the present invention, the concentrations of the Taqman probe and the public beacon probe are both 20nM, the volume ratio is 2:1, and the total volume is 1μL) needs to be added to the reaction system in Table 2, and the 5' end thereof contains a fluorescent reporter group, and the 3' end contains a fluorescent quenching group. Before the fluorescent probe is bound by the probe primer region of the Taqman probe through complementary pairing, the fluorescent probe is in a curled state, and the fluorescent reporter group is quenched by the adjacent fluorescent quenching group and does not emit a fluorescent signal; when the probe primer region binds to the target sequence and begins to extend, the configuration of the fluorescent probe changes, the 5' end and the 3' end are separated, and the fluorescent reporter group begins to emit a signal.

[0076] Figure 5 The following are some results of using the traditional Taqman probe before improvement to detect negative samples. Since the biological sample to be tested is a negative sample extracted with sterile nuclease-free water, no melting peak should appear in the melting curve. Figure 5 As shown in the figure, the low melting peaks in the same Tm region of each channel are actually generated by the background fluorescence signal in the system and are non-specific background peaks. Since the background peak height of some channels (such as FAM) is higher than the positive judgment threshold (8.0), this background peak will cause the channel to misjudge the result as a false positive, interfering with the accuracy of the final diagnosis result.

[0077] When the multiple nucleic acid detection system of Example 1 of the present invention is used to detect negative samples, the results are as follows: Figure 6 As shown. Figure 6 It can be seen that there is no Figure 5 It can be seen that the multiple nucleic acid detection system of the present invention will not have non-specific background peaks, thus will not cause misleading judgments on the results, and the results are more accurate and reliable.

[0078] Comparative Example 2 Effect of the Improved Multiple Nucleic Acid Detection System Combined with the Traditional PCR System on the Detection Results

[0079] This comparative example uses the multiple nucleic acid detection system of Example 1 of the present invention in combination with a traditional PCR system without the addition of ammonium sulfate to detect influenza B virus (FAM83-87°C), Bordetella parapertussis (VIC74-78°C), human metapneumovirus (ROX 79-83°C), and coronavirus (Cy568-72°C) positive standards, and analyzes their melting curves. The target sequences of these four pathogens are selected from the four channels of FAM, VIC, ROX, and Cy5, respectively, and the Tm values ​​of the melting curves of the target sequences do not overlap.

[0080] The positive standard nucleic acid was extracted, and the PCR reaction system was prepared according to Table 5 (traditional PCR system) and Table 2. The PCR reaction was performed according to the process of Table 3 to obtain the PCR reaction product.

[0081] Table 5

[0082] Components Add volume 5×PCRBuffer 5μL DNA polymerase (5U / μL) 5μL dNTPs(20mM) 0.5μL Reverse transcriptase (200 U / μL) 0.5μL UDG enzyme (1U / μL) 0.5μL RNaseInhibitor (40U / μL) 0.5μL Amplification primer mixture (0.8pmol / μL) 1μL Target probe (1.6 pmol / μL) 1μL Public beacon probe (1.0pmo / μL) 1μL <![CDATA[MgCl2]]> 7mM KCl 0.1M Purified water Add water to the reaction system to 20 μL

[0083] The melting curve analysis of each channel of the PCR product was performed to determine the type of target nucleic acid sequence present in the reaction system. The target pathogen was detected using the FAM, VIC, ROX, and Cy5 channels. When there was a melting peak within the Tm reference value range of the specific pathogen and the peak height exceeded 8, the pathogen within the Tm range was determined to be positive according to Table 4; when two or more melting peaks appeared at the same time, it was determined that the sample was infected with two or more pathogens at the same time.

[0084] When the PCR reaction system in Table 5 is used to detect the positive standard nucleic acid, the results are as follows Figure 7 As shown. Figure 7 It can be seen that there are a total of 5 melting peaks belonging to 4 channels, including: Cy571~72℃, ROX81~82℃, FAM86~87℃, VIC76~77℃, and FAM76~77℃, and the corresponding pathogens are: coronavirus, human metapneumovirus, influenza B virus, Bordetella parapertussis and new coronavirus (N gene). However, the selected positive standard sample does not contain the new coronavirus (N gene), and the peak value of the corresponding FAM76~77℃ positive peak is low, so it is suspected that the positive peak of the FAM76~77℃ channel is actually a false positive peak.

[0085] When the PCR reaction system in Table 2 is used to detect the positive standard nucleic acid, the results are as follows Figure 8 As shown. Figure 8It can be seen that there are 4 melting peaks belonging to 4 channels, including: Cy571~72℃, ROX81~82℃, FAM86~87℃, and VIC76~77℃. The corresponding pathogens are: coronavirus, human metapneumovirus, influenza B virus and Bordetella parapertussis, which are completely consistent with the input positive target sequence. Figure 7 The FAM76~77℃ positive peak is shown. Figure 7 The positive peak in the FAM76-77℃ channel is actually a false positive peak. By improving the PCR reaction system, the false positive peak no longer appears.

[0086] Therefore, by using the PCR reaction system of the present invention to perform multiple nucleic acid detection, the problem of false positive interpretation caused by background peaks can be eliminated, while ensuring that no false positive peaks appear in the presence of multi-channel target sequences, making the interpretation of positive results more accurate.

[0087] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0088] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations 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 of the present invention shall be subject to the attached claims.

Claims

1. A detection kit for respiratory diseases, characterized in that: A detection system comprising multiple nucleic acids and (NH4)2SO4; the detection system comprising multiple nucleic acids comprises a probe combination, the probe combination comprises a target probe and a public beacon probe; The Target probe includes, from the 5' end to the 3' end, a probe primer region, a Tag enzyme exonuclease site and a Target region; the 5' end of the probe primer region is modified with a fluorescent quenching 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 quenching group 2; the 3' end of the Target region contains a nucleotide sequence that is reverse complementary to the target nucleic acid sequence; the probe primer region contains a nucleotide sequence that is non-hybridized and complementary to the target nucleic acid sequence; The public beacon probe includes: a 5' terminal region, 10 to 20 primer binding regions and a 3' terminal region in sequence; each of the primer binding regions is separated by a C3 Spacer; the 3' end of each of the primer binding regions has a sequence that can be complementary to the 5' end of the probe primer region of the Target probe; the length and sequence of each of the primer binding regions are different; the length of each of the primer binding regions is 15 to 35 bases; the end of the 3' terminal region is 4 consecutive G bases.

2. The respiratory disease detection kit according to claim 1, characterized in that: The sequence of the Target region of the Target probe is reverse complementary to the target nucleic acid sequence.

3. The respiratory disease detection kit according to claim 1, characterized in that: The 3' end region of the public beacon probe consists of 5 to 8 bases.

4. The respiratory disease detection kit according to claim 1, characterized in that: The fluorescent reporter group, fluorescent quencher group 1 and fluorescent quencher group 2 are all selected from FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas, Red, LCRED640, Cy5, LC RED705, AlexaFluor 488 and Alexa Fluor 750.

5. The respiratory disease detection kit according to claim 4, characterized in that: The fluorescence quenching group 1 and the fluorescence quenching group 2 are the same or different.

6. The respiratory disease detection kit according to any one of claims 1 to 4, characterized in that: The multiple nucleic acid detection system also includes amplification primers for amplifying target nucleic acid sequences.

7. The respiratory disease detection kit according to claim 6, characterized in that: The sequence length of the 5' end of the amplification primer is 18 to 25 bp; the sequence length of the 3' end of the amplification primer is 10 to 15 bp.

8. A method for detecting multiple nucleic acids for non-diagnostic purposes, characterized in that: Using the detection kit described in claim 6 or 7, a PCR reaction is performed on the test sample, and then a melting curve analysis is performed; in the reaction system of the PCR reaction, the concentration of the Target probe is 1.0pmol / μL~3.0pmol / μL, the concentration of the amplification primer is 0.3pmol / μL~2.0pmol / μL, the concentration of the public beacon probe is 0.2pmol / μL~2.2pmol / μL, and the working concentration of (NH4)2SO4 is 0.01~0.2M.

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