Reagent, kit and method for detecting multiple nucleic acids

Abstract

A reagent, kit and method for detecting multiple nucleic acids. The reagent comprises an amplification primer for amplifying a target nucleic acid sequence and a Taqman probe. The Taqman probe is composed of a probe primer region, a Taq enzyme exonuclease site, a Target region, and a probe beacon region, sequentially from the 5' end to the 3' end, wherein the sequence of the Target region is reversely complementary to the target nucleic acid sequence, and fluorescent quenching group 1 is connected to the 3' end of the Target region; and a fluorescent reporter group is connected to the 5' end of the probe beacon region, fluorescent quenching group 2 is connected to the 3' end of the probe beacon region, and the probe beacon region comprises a sequence that is reversely complementary to and paired with the probe primer region. The reagent, when used for detection, exhibits the significant advantages of no non-specific background peaks and no bimodal / wide peaks and can simultaneously detect a plurality of target sequences more accurately.

Description

Reagent, kit and method for detecting multiple nucleic acids TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a reagent, kit and method for detecting multiple nucleic acids. BACKGROUND

[0002] There are various detection technology routes in the field of nucleic acid detection, including probe molecule hybridization, nucleic acid-protein interaction and sequencing technology. Among them, PCR based on the amplification principle of target sequences is the most widely used technology platform. The process of PCR in vitro amplification of nucleic acids includes the cyclic repetition of "denaturation of DNA double strands-primer annealing and binding-primer extension".

[0003] At present, the PCR platform has been continuously improved and developed, and RT-PCR technology using reverse transcriptase to reverse transcribe RNA in the sample into cDNA for amplification, real-time fluorescent quantitative PCR technology using fluorescent reporter molecules to detect amplification products in real time, and digital PCR technology for accurate quantification have appeared. Under the premise of meeting the detection needs of multiple targets, in order to improve efficiency and control economic and time costs, the multiplex PCR technology for detecting multiple targets in a single tube reaction system has attracted widespread attention.

[0004] The target sequence detection methods based on nucleic acid amplification mainly include:

[0005] 1. The amplification products are distinguished according to their different sizes by polyacrylamide gel electrophoresis to achieve the purpose of identification. The problem is that the process is slow and inefficient, and needs manual operation, and is easy to be contaminated.

[0006] 2. Molecular beacon method based on fluorescence resonance energy transfer (FRET, fluorescence resonance energy transfer) principle. The beacon molecule forms a hairpin structure in the liquid system, and the two dyes are adjacent and do not occur FRET. When the beacon hybridizes with the target sequence, the fluorescence and quencher group are separated. Problem: there is a hairpin structure that can match the target sequence.

[0007] 3. Use hybridization and nuclease activity. Taqman probe will bind to the middle of the amplification region before the extension of the amplification primer, and use the 5' exonuclease activity of polymerase to cut the probe, release the fluorescence group and quencher group on the probe. For multiplex real-time fluorescent PCR based on Taqman and molecular beacon combination, different fluorescence groups are used to label the oligonucleotide probes specific to the target sequence. By detecting the fluorescence signals possessed by different probes, the target sequences specifically recognized by each probe can be detected. However, due to the limitation of the types of fluorescence labels and the number of device channels, the number of multiple detection targets is also limited, so that the number of detection targets in a single tube is generally not more than 6.

[0008] 4. Melting curve method. In real-time fluorescent quantitative PCR, the target sequence can be detected by two modes, real-time detection in the amplification stage and melting curve analysis after amplification. In the melting curve stage, the probe sequence of the primer probe hybridizes with the target sequence to form a double strand, and during the extension of the target sequence, the Taqman enzyme will cut off the primer end of the primer probe. At a certain temperature, the free primer end binds with the molecular beacon with a fluorescent group and a quencher group and opens the extension, thereby producing a positive peak in the melting curve for identifying the presence of the target sequence. However, the melting curve method has the following limitations:

[0009] (1) The molecular beacon is in a free state, and the quencher group attached thereto will close the fluorescent group attached thereto when the molecular beacon is in a curled state, but under the PCR reaction condition, the non-specific binding with part of the sequence will cause incomplete fluorescence closure, thereby affecting the accuracy and precision of the analysis result.

[0010] (2) The free molecular beacon can bind with the primer end of the uncut probe primer to form a double strand (but cannot be extended), thereby having a probability of affecting the cutting position of the Taqman enzyme when the probe end binds with the target sequence, resulting in a difference of 1-3 bp in the length of the cutting product, i.e., the free probe primer, from the preset length. The length difference appears randomly and can affect the extension temperature of the probe primer after binding with the probe beacon, thereby causing a double peak / wide peak phenomenon in the final melting curve (if the Tm values of the two peaks in the double peak are close, a relatively flat wide peak phenomenon will be generated), affecting the accuracy and precision of the analysis result.

[0011] Therefore, it is necessary to provide a more accurate and precise method for detecting multiple nucleic acids to solve the problems of non-specific peaks and double peaks / wide peaks.

[0012] SUMMARY

[0013] Based on this, the purpose of the present application is to provide a reagent, kit and method for detecting multiple nucleic acids, which can improve the accuracy of detecting target sequences by using the detection reagent of the present application.

[0014] The specific technical solutions for achieving the above-mentioned application purposes include the following.

[0015] In a first aspect of the present application, a reagent for detecting multiple nucleic acids is provided, which comprises an amplification primer and a Taqman probe for amplifying a target nucleic acid sequence; wherein,

[0016] The Taqman probe comprises, from 5' end to 3' end, a probe primer region, a Tag exonuclease site, a Target region, and a probe beacon region;

[0017] The sequence of the Target region is reverse complement to the target nucleic acid sequence, and a fluorescence quenching group 1 is connected to the 3' end of the Target region;

[0018] The 5' end of the probe beacon region is connected with a fluorescence reporter group, and the 3' end is connected with a fluorescence quenching group 2; the probe beacon region comprises a sequence which is reverse complement to the primer region of the probe.

[0019] In a second aspect of the present application, a kit for detecting multiple nucleic acids is provided, comprising the reagent for detecting multiple nucleic acids described above.

[0020] In a third aspect of the present application, a method for detecting multiple nucleic acids is provided, comprising the following steps:

[0021] (1) designing the reagent for detecting multiple nucleic acids described above according to the pathogen to be detected;

[0022] (2) mixing the nucleic acid of the biological sample to be detected, DNA polymerase and the reagent for detecting multiple nucleic acids described in step (1) to prepare a PCR reaction system, and performing PCR reaction and melting curve analysis.

[0023] The present application has the following beneficial effects:

[0024] In the present application, the structure of Taqman probe is optimized, on the one hand, the Taqman probe adopts a double fluorescence quenching structure, and on the other hand, the sequence of the probe beacon region and the probe primer region can be complementary to each other to form a double strand, which will form a self-sealing structure when off-target. Under this concept, when the reagent for detecting multiple nucleic acids of the present application is used for detection, it has the remarkable advantages of no non-specific background peak and no double peak / broad peak phenomenon, and multiple target sequences can be more accurately detected simultaneously. BRIEF DESCRIPTION OF DRAWINGS

[0025] Fig. 1 is a structural schematic diagram of the Taqman probe in the present application.

[0026] Figs. 2-5 are working principle diagrams of the reagent for detecting multiple nucleic acids of the present application.

[0027] Fig. 6 is a partial melting curve result of detecting a negative sample using the Taqman probe before improvement in the comparative example 1 of the present application.

[0028] Fig. 7 is a partial melting curve result of detecting a negative sample using the Taqman probe of the present application in the comparative example 1 of the present application.

[0029] Fig. 8 is a partial melting curve result of detecting a clinical sample using the Taqman probe before improvement in the comparative example 1 of the present application; from left to right, the melting curves of FAM, HEX and ROX channels are shown.

[0030] Figure 9 is a partial melting curve result of detecting clinical samples by using the Taqman probe of the present application in Example 1 of the present application; from left to right are the melting curves of FAM, HEX and ROX channels.

[0031] Figure 10 is a melting curve of detecting qualified positive quality control by using the existing "ten respiratory tract pathogen nucleic acid detection kit" in Example 2 of the present application.

[0032] Figure 11 is a melting curve of detecting qualified positive quality control by using the multiple detection nucleic acid detection reagent of the present application in Example 2 of the present application. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of the present application, the present application will be described in more detail below. The present application can be realized 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 disclosure of the present application more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used in the present application includes any and all combinations of one or more related listed items.

[0035] Please refer to Figure 1, which is a structural schematic diagram of the Taqman probe in the multiple detection nucleic acid detection reagent of the present application. Please refer to Figures 2-5, which can be used to understand the detection principle of the multiple detection nucleic acid detection reagent of the present application in detail (the probe primer in Figures 1-5 refers to the same sequence as the probe primer in this paper, and the probe beacon in Figures 1-5 refers to the same sequence as the probe beacon in this paper):

[0036] In the PCR amplification process of the target sequence (DNA or nucleic acid mixture, the target sequence herein can be exchanged with the target nucleic acid sequence, which points to the same sequence), if the sequence of the Target region of the Taqman probe can be combined with the target sequence by complementary pairing (Figure 2), when the target nucleic acid sequence to be amplified is extended to the Tag enzyme exonuclease site of the Taqman probe, the Taqman enzyme will cut the connection position of the probe primer region and the Target region (Tag enzyme exonuclease site), so that the probe primer region and the Target region are separated and free; with the extension, the Target region combined with the target sequence is gradually removed, and the probe beacon region is also free (Figure 3). When the probe primer region is not combined, the free probe beacon region is in a curled state, the 5' end fluorescent reporter group and the 3' end fluorescent quencher 2 (Quencher2) are close to each other, and no fluorescent signal is generated; when the free probe primer region combines with the free probe beacon region and begins to extend, the probe beacon region will be unfolded during the extension, so that the terminal fluorescent reporter group generates a fluorescent signal (Figure 4). With the PCR amplification of the target sequence to the platform, the fluorescent signal accumulates to a certain value and no longer increases, so the amplification curve of the target sequence in this channel can be made. Because the length of the probe primer region or the probe beacon region is different from the target sequence, the melting curve of the combined extension product is also different. Ensuring that the extension product of the probe beacon region with different melting temperatures and the target sequence complementary to the Target region correspond one by one, so that the presence of different melting peaks corresponds to the target sequence.

[0037] If the Target region of the Taqman probe fails to combine with the target sequence by complementary pairing (Off-Target), the probe primer region will recognize the probe beacon region combined with the Taqman probe itself to form a self-sealing state (Figure 5). Because the probe primer region is not exonucleolyzed, the probe primer region will not be extended after combining with the Target region. At the same time, because the Taqman probe will not be free with the extension of the target sequence, the fluorescent reporter group at the 5' end of the probe beacon region is always close to the fluorescent quencher 1 (Quencher1) at the 3' end of the Target region in the quenching state, so there will be no non-specific background signal.

[0038] When the Target region of the Taqman probe combines with the target sequence by complementary pairing (On-Target), the extension of the upstream primer will make the Target region complementary to the target sequence gradually removed by the Taqman enzyme; at this time, the self-sealing structure of the Taqman probe will make the Taqman enzyme accurately cut the connection position of the probe primer region and the Target region, ensuring that the cutting product, i.e., the free probe primer region, has a stable expected length, thereby avoiding the appearance of double peaks / broad peaks.

[0039] The inventor of the present application develops a more efficient, convenient and accurate reagent for detecting multiple nucleic acids based on in-depth research on gene detection technology, which comprises amplification primers for amplifying target nucleic acid sequences and Taqman probes, and the structure of the Taqman probe is optimized. On the one hand, the Taqman probe adopts a double fluorescence quenching structure, which ensures that the fluorescence reporter group is always in a quenched state before the Taqman probe is combined with the target sequence, so that false positive peaks or non-specific background peaks will not appear. On the other hand, when the Taqman probe is not combined with the target sequence, the probe beacon area and the probe primer area will form a double-stranded structure due to sequence complementarity, and a self-sealing structure will be formed when the target sequence is not detected (because of the existence of the double fluorescence quenching structure, the fluorescence group in the probe beacon area will not produce a fluorescence signal at this time), which further maintains the stability of the structure. When the Taqman probe in this state is combined with the target sequence, it can ensure that the Taqman enzyme accurately cuts the connection position between the probe primer area and the Target area, and ensure that the length of the cutting product, i.e. the free probe primer area, is stable at the expected value, thereby preventing the appearance of double peaks / wide peaks in the melting curve. At the same time, by keeping the sequence of the probe primer area consistent and adjusting the sequence and length of the probe beacon area, or keeping the sequence of the probe beacon area consistent and adjusting the sequence and length of the probe primer area, the combination and extension product of the probe primer area and the probe beacon area has a unique melting curve, which is a single curve positive peak, and the spacing between the peaks is appropriate. Without the need for post-processing of experimental data, the situation of missed detection or multiple detection is avoided to the greatest extent. Therefore, the reagent for detecting multiple nucleic acids of the present application can realize accurate and high-sensitivity simultaneous detection of multiple target sequences.

[0040] In some embodiments of the present application, a reagent for detecting multiple nucleic acids is disclosed, which comprises amplification primers for amplifying target nucleic acid sequences and Taqman probes.

[0041] The Taqman probe comprises, from 5' end to 3' end: a probe primer area, a Tag enzyme excision site, a Target area, and a probe beacon area.

[0042] The sequence of the Target area is reverse complementary to the target nucleic acid sequence, and the 3' end of the Target area is connected with a fluorescence quenching group 1.

[0043] The 5' end of the probe beacon area is connected with a fluorescence reporter group, and the 3' end is connected with a fluorescence quenching group 2; the probe beacon area comprises a sequence that is reverse complementary to the probe primer area.

[0044] In some embodiments, the fluorescent quenching group 1 is connected at a distance of 1-20 bp from the 3' end of the Target region 3; the fluorescent reporter group is connected at a distance of 1-5 bp from the 5' end of the probe beacon region; and the fluorescent quenching group 2 is connected at a distance of 1-20 bp from the 3' end of the probe beacon region. When the Target region is not cleaved and separated from the probe beacon region, the fluorescent reporter group emits fluorescence which is quenched by the fluorescent quenching group 1; when the probe beacon region is free but not complementary to the probe primer region and extended, the probe beacon region is in a crimped state, the fluorescent reporter group emits fluorescence which is quenched by the fluorescent quenching group 2.

[0045] In some embodiments, the length of the Taqman probe is 60-142 bp; the length of the probe primer region is 10-30 bp; the length of the Target region is 30-72 bp; and the length of the probe beacon region is 20-40 bp.

[0046] In some embodiments, the length of the primer region of the probe is 15-20 bp; the length of the Target region is 45-70 bp; and the length of the probe beacon region is 22-37 bp.

[0047] In some embodiments, the fluorescent reporter group is FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas, Red, LC RED640, Cy5, LC RED705, Alexa Fluor 488 or Alexa Fluor 750.

[0048] In some embodiments, the fluorescent quenching group 1 is the same as or different from the fluorescent quenching group 2.

[0049] In some embodiments, the amplification primer includes an upstream primer and a downstream primer, and the length of the upstream primer and the downstream primer is 18-25 bp. The length of the amplification product of the amplification primer is 80-200 bp, and the amplification product contains the Target region in the Taqman probe which is complementary to the target sequence and is spaced apart from the amplification primer by 3-50 bp.

[0050] In some embodiments of the present application, a kit for detecting multiple nucleic acids is provided, which includes the reagent for detecting multiple nucleic acids.

[0051] In some embodiments of the present application, a method for detecting multiple nucleic acids is provided, which includes the following steps:

[0052] (1) designing the reagent for detecting multiple nucleic acids according to the pathogen to be detected;

[0053] (2) mixing the nucleic acid of the biological sample to be tested, the DNA polymerase and the reagent for detecting the plurality of nucleic acids in step (1) to prepare a PCR reaction system, and performing PCR reaction and melting curve analysis.

[0054] In some embodiments, the working concentration of the amplification primer in the PCR reaction system is 0.7 pmol / μL to 0.9 pmol / μL, and the working concentration of the Taqman probe is 1.5 pmol / μL to 1.7 pmol / μL.

[0055] In some embodiments, the Taqman probe comprises a probe group 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 than 20 target nucleic acid sequences.

[0056] In some embodiments, the amplification primer comprises a primer pair 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 than 20 target nucleic acid sequences.

[0057] In some embodiments, the pathogen is a pathogen of respiratory infection, gastrointestinal 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.

[0058] In some embodiments, the biological sample to be tested can be selected from the group consisting of, 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, viral culture, cell line culture, artificially synthesized plasmid sample.

[0059] In some embodiments, the nucleic acid of the biological sample to be tested 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.

[0060] The present application will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0061] Example 1 Multiplex nucleic acid detection reagent and detection method

[0062] The present embodiment provides a method for detecting pathogens of a biological sample to be tested using a multiplex nucleic acid detection reagent, comprising the following steps:

[0063] I. Designing a multiplex nucleic acid detection reagent

[0064] 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 multiplex nucleic acid detection reagent (including amplification primer and Taqman probe) is designed. The sequences of the upstream primer F n , the downstream primer R n , the Target region P n of the Taqman probe, and the entire Taqman probe are shown in Table 1.

[0065] Table 1 Note: In Table 1, Pn is the Target region of the Taqman probe, the primer region before Pn is the probe primer region, and the primer region after Pn is the probe beacon region. The underlined part is the sequence complementary to the probe primer region.

[0066] II. Obtain the nucleic acid of the biological sample to be tested;

[0067] III. Mix the nucleic acid of the biological sample to be tested, DNA polymerase and the multiplex nucleic acid detection reagent of Table 1 to prepare a PCR reaction system and perform PCR reaction.

[0068] The PCR reaction system is shown in Table 2, and the PCR reaction program is shown in Table 3.

[0069] Table 2

[0070] Table 3

[0071] IV. Melting curve analysis

[0072] The types of target nucleic acid sequences 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.

[0073] Table 4

[0074] Influence of traditional Taqman probe and Taqman probe of the application on detection results

[0075] In the present comparative example, a negative sample (sterile nucleic acid extracted from water) and three clinical pharyngeal swab samples (i.e. biological samples to be tested) are detected by using a multiplex nucleic acid detection reagent containing a traditional Taqman probe and a multiplex nucleic acid detection reagent containing a Taqman probe of the application, respectively. The PCR reaction system is prepared according to Table 2, 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 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, it is determined that the pathogen in the Tm range is 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 infected with two or more pathogens at the same time, and then the types of pathogens present in the biological sample to be tested are determined.

[0076] The traditional Taqman probe includes a probe primer region and a Target region, but does not include a probe beacon region and does not 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 Taqman probe of the application. When the multiplex nucleic acid detection reagent containing the traditional Taqman probe is used for detection, a fluorescent probe (equivalent to a molecular beacon, the concentration of the Taqman probe and the molecular beacon is 20 nM, the volume ratio is 2:1, and the total volume is 1 μL) for melt curve analysis needs to be additionally added in the reaction system of Table 2. The 5' end of the fluorescent probe contains a fluorescent reporter group, and the 3' end contains a fluorescent quencher group. Before the fluorescent probe is combined 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 fluorescent quencher group close to it and does not emit a fluorescent signal. When the probe primer region of the Taqman probe 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.

[0077] I. Negative sample

[0078] Since the biological sample to be tested is a negative sample for nucleic acid extraction with sterile nucleic acid-free water, there should be no melting peak in the melting curve. If low melting peaks appear in the same Tm range in each channel, they are actually caused by background fluorescence signals in the system and are non-specific background peaks.

[0079] Figure 6 shows partial results of detecting a negative sample using the improved reagent for detecting multiple nucleic acids. As shown in Figure 6, the background peak height of some channels (such as FAM) is higher than the positive determination threshold (8), which may cause false positive misjudgment of the results by the channel. Therefore, using the traditional Taqman probe, non-specific background peaks may appear during melting curve analysis due to the lack of double-quenching group structure in the design.

[0080] When the reagent for detecting multiple nucleic acids of the present application is used to detect a negative sample, the results are shown in Figure 7. As shown in Figure 7, no melting peak appears in the melting curve of each channel. It can be seen that the reagent for detecting multiple nucleic acids of the present application does not produce non-specific melting peaks and does not cause false positive misjudgment of the results, so the results are more accurate and reliable.

[0081] II. Clinical samples

[0082] Figure 8 shows partial results of detecting three clinical pharyngeal swab samples using the improved reagent for detecting multiple nucleic acids. As shown in Figure 8, the three clinical samples each produce a positive peak in one Tm range in one channel, and the corresponding pathogens are: novel coronavirus (FAM 72-74℃), Chlamydia pneumoniae (VIC 85-86℃), and human metapneumovirus (ROX 81-82℃). However, the melting peaks produced by the three channels all show double peaks / wide peaks, i.e. another positive peak appears within a certain range from the highest main peak. The double peaks produce multiple peak values for the positive peak, and the wide peaks produced by the combination of the double peaks that are too close increase the Tm value range corresponding to the peak value, both of which may affect the final result determination, especially causing serious interference with the automatic interpretation of the instrument. Therefore, using the traditional Taqman probe, double peaks or wide peaks may appear during melting curve analysis.

[0083] When the reagent for detecting multiple nucleic acids of the embodiment 1 of the present application was used to detect three clinical collected throat swab samples, the results were shown in Figure 9. As can be seen from Figure 9, the three clinical samples produced positive peaks in only one Tm interval of one channel, and the corresponding pathogens were: novel coronavirus (FAM 72-74℃), Chlamydia pneumoniae (VIC 85-86℃), and human metapneumovirus (ROX 81-82℃). The melting curves of the three channels were all typical single positive peaks, and no double peak / broad peak phenomenon occurred. The instrument's automatic interpretation module also gave the interpretation results of novel coronavirus, Chlamydia pneumoniae and human metapneumovirus. It can be seen that the reagent for detecting multiple nucleic acids of the present application does not have the double peak / broad peak phenomenon, and the results are more accurate and reliable.

[0084] Example 2: Detection of virus positive standard by using existing respiratory tract pathogen nucleic acid detection kit

[0085] In this comparative example, the "ten respiratory tract pathogen nucleic acid detection kit" produced by X company (of which the ten pathogens are coronavirus, rhinovirus, human metapneumovirus, adenovirus, novel coronavirus, influenza A virus, influenza B virus, respiratory syncytial virus, Bordetella pertussis, and parapertussis Bordetella) was used to detect the positive standard of coronavirus, rhinovirus and human metapneumovirus, wherein rhinovirus A2 was from Jingliang Technology (Shenzhen) Co., Ltd., coronavirus OC43 was from China Center for Type Culture Collection, and human metapneumovirus B2 was from Jingliang Technology (Shenzhen) Co., Ltd. The results were shown in Figure 10.

[0086] As can be seen from Figure 10, a total of five melting peaks belonging to four channels were produced, including: Cy5 70-72℃, ROX 71-73℃, ROX 77-80℃, FAM 73-74℃, and VIC 73-74℃, and the corresponding pathogens were: coronavirus, rhinovirus, adenovirus or human metapneumovirus, novel coronavirus and Bordetella pertussis. However, the melting peak of the ROX channel at the position of 77-80℃ was a broad peak, and the peak value corresponded to a large range of Tm values, making it difficult to determine whether the result was adenovirus (ROX 74-78℃) or human metapneumovirus (ROX 79-84℃). In addition, the positive peaks of the FAM and VIC channels at 73-74℃ had low peak values, and the peak values of the two channels were consistent with the Tm value position and the peak type height of the positive peak, so it was suspected that the positive peaks of the FAM and VIC channels were actually non-specific background peaks.

[0087] When the reagent for detecting multiple nucleic acids of the embodiment 1 of the present application is used to detect the above-mentioned virus positive standard sample, the result is shown in Figure 11. As can be seen from Figure 11, there are totally 3 melting peaks belonging to 2 channels, including: Cy5 70-72℃, ROX 71-73℃, ROX 79-80℃, corresponding to the pathogens: coronavirus, rhinovirus and human metapneumovirus, which are consistent with the selected positive quality control. Among them, the positive peak of the ROX channel at the position of 79-80℃ is typical, and the double peak / broad peak phenomenon does not occur, thereby not affecting the interpretation of the detection result (ROX 79-84℃ is human metapneumovirus); in addition, there is no positive peak in the FAM and VIC channels, which proves that the positive peaks in the FAM and VIC channels in Figure 10 are non-specific background peaks, and the non-specific background peaks do not appear in any channel in Figure 11.

[0088] Therefore, the improved reagent for detecting multiple nucleic acids of the present application eliminates the problem of false positive interpretation caused by background peaks, and at the same time ensures the typicality of the positive peak shape, so that the interpretation of the positive result is more accurate.

[0089] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description simple, all possible combinations of the technical features in the above-mentioned embodiments are not described, but as long as the combination of the technical features does not exist contradictory, it should be considered as the scope of the present application.

[0090] The above-mentioned embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary 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 protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A reagent for detecting multiple nucleic acids, characterized by comprising: The reagent comprises an amplification primer and a Taqman probe for amplifying a target nucleic acid sequence; wherein The Taqman probe comprises, from 5' end to 3' end, a probe primer region, a Tag exonuclease site, a Target region, and a probe beacon region; The sequence of the Target region is reverse complementary to the target nucleic acid sequence, and the 3' end of the Target region is connected with a fluorescence quenching group 1; The 5' end of the probe beacon region is connected with a fluorescence reporter group, and the 3' end is connected with a fluorescence quenching group 2; the probe beacon region comprises a sequence reverse complementary to the probe primer region.

2. The reagent for detecting multiplex nucleic acids according to claim 1, wherein The fluorescence quenching group 1 is connected at a distance of 1-20 bp from the 3' end of the Target region; and / or The fluorescence reporter group is connected at a distance of 1-5 bp from the 5' end of the probe beacon region; and / or The fluorescence quenching group 2 is connected at a distance of 1-20 bp from the 3' end of the probe beacon region.

3. The reagent for detecting multiplex nucleic acids according to claim 1, wherein The length of the Taqman probe is 60-142 bp; The length of the probe primer region is 10-30 bp; the length of the Target region is 30-72 bp; and the length of the probe beacon region is 20-40 bp. The length of the probe primer region is 15-20 bp; the length of the Target region is 45-70 bp; and the length of the probe beacon region is 22-37 bp.

4. The reagent for detecting multiplex nucleic acids according to claim 3, wherein The fluorescence reporter group is FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas, Red, LC RED640, Cy5, LC RED705, Alexa Fluor 488, or Alexa Fluor 750.

5. The reagent for detecting multiple nucleic acids according to any one of claims 1 to 4, wherein The fluorescence quenching group 1 is the same as or different from the fluorescence quenching group 2.

6. The reagent for detecting multiple nucleic acids according to any one of claims 1 to 4, wherein The amplification primer comprises an upstream primer and a downstream primer, and the length of each of the upstream primer and the downstream primer is 18-25 bp.

7. The reagent for detecting multiple nucleic acids according to any one of claims 1 to 4, wherein The reagent for detecting multiple nucleic acids according to any one of claims 1-7.

8. A kit for detecting multiple nucleic acids, characterized by, The method comprises the following steps:

9. A method of detecting multiple nucleic acids, characterized by, (1) designing the reagent for detecting multiple nucleic acids according to any one of claims 1-7 for a pathogen to be detected; (2) mixing the nucleic acid of a biological sample to be detected, DNA polymerase, and the reagent for detecting multiple nucleic acids of step (1) to prepare a PCR reaction system, and performing PCR reaction and melting curve analysis. In the PCR reaction system, the working concentration of the amplification primer is 0.7 pmol / μL-0.9 pmol / μL, and the working concentration of the Taqman probe is 1.5 pmol / μL-1.7 pmol / μL.

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. ​

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