Method for detecting multiple target nucleic acids based on digital PCR (Polymerase Chain Reaction)

Abstract

On the basis of six-channel digital PCR and by adopting a non-stem-loop type general report probe and a detection probe, reagents such as a multi-target nucleic acid primer, the detection probe and the general report probe are added into a single reaction hole for amplification, and a special analysis method is adopted. According to the method, the number of detection targets can be increased to 15 only by using a general reporter probe marked by six different fluorophores with fixed sequences in a single hole, so that the phenomenon of fluorescence crosstalk between different fluorescence signals when multiple target nucleic acid detection is carried out by a TaqMan probe method is eliminated; the type and the number of fluorescent groups modified on each to-be-detected target nucleic acid recognition probe in a TaqMan probe method are greatly reduced, and the detection cost is reduced.

Description

Technical Field

[0001] The present application relates to multiplex detection of nucleic acid molecules. The present application provides a method for multiplex target nucleic acid detection based on digital PCR, which can quantitatively detect multiple target nucleic acid sequences in a single-well reaction system. Background Art

[0002] The multiplex fluorescence quantitative PCR developed in recent years has concentrated the detection of multiple target nucleic acids into one reaction tube. This method not only saves the use of precious samples and reduces the detection cost, but also has the accuracy and sensitivity of fluorescence quantitative PCR. However, the maximum number of target nucleic acid sequences that can be detected by this method depends on the number of channels of the fluorescence quantitative PCR instrument and the influence of crosstalk between fluorescent signals. Therefore, the number of targets that can be detected in one reaction tube is still small. The digital PCR detection method is an absolute quantitative nucleic acid detection method, and attempts are currently being made to detect multiple target nucleic acids in a single reaction well. However, in the application of the TaqMan probe method as the main detection method, if there are more than six target nucleic acids in a single reaction well, the detection results will also be limited to the number of fluorescence detection channels of the instrument and the influence of crosstalk between fluorescent signals. Therefore, it is very important to develop a new digital PCR method that is different from the TaqMan probe method as a single reaction well multi-target nucleic acid detection method. It can not only save samples and reduce costs, but also accurately quantify multiple target nucleic acids. Summary of the invention

[0003] Based on this, it is necessary to develop a new method mainly for digital PCR detection of multiple target nucleic acids in a single reaction well.

[0004] The present application provides a method for multi-target nucleic acid detection based on digital PCR, comprising the following main steps:

[0005] (1) Provide the following test components:

[0006] (a) For each target nucleic acid to be detected, an upstream primer, a downstream primer and a detection probe are provided; the detection probe is composed of three parts from 5' to 3', including a tag sequence, a complementary sequence to a partial sequence of the target nucleic acid, and a blocking sequence; wherein the 3' end of the blocking sequence or the interior of the sequence is labeled with one or more fluorescent quenching groups; and when the detection probe hybridizes with the target nucleic acid sequence, the upstream primer is located upstream of the detection probe;

[0007] (b) providing one or more universal reporter probes for each target nucleic acid to be detected; wherein each universal probe is independently composed of two parts from the 5' to the 3' direction, including a complementary sequence to the blocking sequence described in (a) and a complementary sequence to the label sequence; and each universal reporter probe is independently labeled with a different fluorescent group at the 5' end;

[0008] Wherein, the sequence composition of the universal reporter probe and the fluorescent group labeled at the 5' end are at least one of the following:

[0009] 5'-FAM-(blocking sequence-tag sequence 1-tag sequence 2-tag sequence 3-tag sequence 4-tag sequence 5) complementary sequence-3';

[0010] 5'-HEX-(blocking sequence-tag sequence 1-tag sequence 6-tag sequence 7-tag sequence 8-tag sequence 9) complementary sequence-3';

[0011] 5'-TAMRA-(blocking sequence-tag sequence 2-tag sequence 6-tag sequence 10-tag sequence 11-tag sequence 12) complementary sequence-3';

[0012] 5'-ROX-(blocking sequence-tag sequence 3-tag sequence 7-tag sequence 10-tag sequence 13-tag sequence 14) complementary sequence-3';

[0013] 5'-Cy5-(blocking sequence-tag sequence 4-tag sequence 8-tag sequence 11-tag sequence 13-tag sequence 15) complementary sequence-3';

[0014] 5'-Cy5.5-(blocking sequence-tag sequence 5-tag sequence 9-tag sequence 12-tag sequence 14-tag sequence 15) complementary sequence-3';

[0015] The blocking sequence, tag sequence and their complementary sequences are shown in the following table:

[0016]

[0017]

[0018] Wherein, the sequence of the universal reporter probe is shown in the following table:

[0019]

[0020] The nucleotide sequences of tag sequence 1 to tag sequence 15 in the optimized universal reporter probe sequence composition shown in the above table have common bases or are added with 1-2 additional bases.

[0021] (2) preparing a sample containing the target nucleic acid sequence to be detected into a digital PCR reaction system and contacting it with the components described in (1) in a single digital PCR chip well, and running a digital PCR reaction program;

[0022] (3) After the digital PCR run is completed, the fluorescence type and fluorescence intensity value of the amplified product are analyzed; the analysis of the fluorescence type and fluorescence intensity value includes the fluorescence signal from the fluorescent marker group described in (1)(b), and the corresponding target nucleic acid sequence is determined based on the measured fluorescence signal type of the fluorescent marker group.

[0023] In certain embodiments, for the detection method of six-channel digital PCR, the number of detection probes can be at least 1, at least 2, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0024] In certain embodiments, for the detection method of six-channel digital PCR, the number of universal reporter probes can be 1, 2, 3, 4, 5 or 6.

[0025] Step (1)

[0026] Detection probe

[0027] like Figure 1 As shown, the sequence of the detection probe is composed of three parts from 5' to 3': a label sequence, a complementary sequence of a partial sequence of the target nucleic acid, and a blocking sequence, wherein the 3' end of the blocking sequence is labeled with one or more and only labeled with a fluorescent quenching group. Therefore, the detection probe is also only labeled with a fluorescent quenching group, and no fluorescent reporter group is labeled. In certain embodiments, the fluorescent quenching group labeled at the 3' end of the blocking sequence of the detection probe or inside the sequence is a molecule or group BHQ-1 or BHQ-2 that can absorb / quench the fluorescence;

[0028] Universal reporter probe

[0029] like Figure 1 As shown, the sequence of the universal reporter probe from 5' to 3' direction consists of two parts: a complementary sequence of the blocking sequence and a complementary sequence of the tag sequence. The 5' end of the universal reporter probe is labeled with a fluorescent reporter group, and the fluorescent reporter group is any suitable group or molecule known in the art, and its specific examples include but are not limited to the fluorescent group labeled at the 5' end of the universal reporter probe described in step (1)(b) is any one of FAM, HEX, TAMRA, ROX, Cy5, and Cy5.5.

[0030] Among them, the sequence composition of the optimized universal reporter probe is as follows:

[0031] 5'-FAM-(blocking sequence-tag sequence 1-tag sequence 2-tag sequence 3-tag sequence 4-tag sequence 5) complementary sequence-3';

[0032] 5'-HEX-(blocking sequence-tag sequence 1-tag sequence 6-tag sequence 7-tag sequence 8-tag sequence 9) complementary sequence-3';

[0033] 5'-TAMRA-(blocking sequence-tag sequence 2-tag sequence 6-tag sequence 10-tag sequence 11-tag sequence 12) complementary sequence-3';

[0034] 5'-ROX-(blocking sequence-tag sequence 3-tag sequence 7-tag sequence 10-tag sequence 13-tag sequence 14) complementary sequence-3';

[0035] 5'-Cy5-(blocking sequence-tag sequence 4-tag sequence 8-tag sequence 11-tag sequence 13-tag sequence 15) complementary sequence-3';

[0036] 5'-Cy5.5-(blocking sequence-tag sequence 5-tag sequence 9-tag sequence 12-tag sequence 14-tag sequence 15) complementary sequence-3';

[0037] Among them, the closed sequence, the tag sequence and the complementary sequence of the tag sequence are shown in the following table:

[0038] sequence name Sequence (5'-3') Complementary sequence (5'-3') Closed Tags TCCTTGGCACC GGTGCCAAGGA Tag sequence 1 gcaCGGCCAACTAACAC GTGTTAGTTGGCCGtgc Tag sequence 2 caGCGTCGCTGAGGCC GGCCTCAGCGACGCtg Tag sequence 3 ctcTACGAGCCAGCGT ACGCTGGCTCGTAgag Tag sequence 4 aggACGACCTGTACGAG CTCGTACAGGTCGTcct Tag sequence 5 cgaTAGACACCGACGAC GTCGTCGGTGTCTAtcg Tag sequence 6 gctCCAATTGCCATAGAC GTCTATGGCAATTGGagc Tag sequence 7 ctcTTATTACTGCTGCTC GAGCAGCAGTAATAAgag Tag sequence 8 gagTCTTGAATGAGTAAAGG CCTTTACTCATTCAAGActc Tag sequence 9 gacAAAGCGTTGCTTTCGA TCGAAAGCAACGCTTTgtc Tag sequence 10 cctCGCTATCGTCACGTG CACGTGACGATAGCGagg Tag sequence 11 ctcCACGTGACGGCATAAC GTTATGCCGTCACGTGgag Tag sequence 12 gccTGACGGCATAACGAC GTCGTTATGCCGTCAggc Tag sequence 13 agccTCCGCACCACCTC GAGGTGGTGCGGAggct Tag sequence 14 attcACTGTCCTACAGCC GGCTGTAGGACAGTgaat Tag sequence 15 agtcgGCATAACGACTATTC GAATAGTCGTTATGCcgact

[0039] Among them, the sequence composition of the universal reporter probe is shown in the following table:

[0040]

[0041]

[0042] Among them, the nucleotide sequences of tag sequence 1 to tag sequence 15 in the sequence composition of the optimized universal reporter probe in the above table have common bases or are added with additional 1-2 bases.

[0043] How detection probes and universal reporter probes work:

[0044] 1. When PCR amplification reaction is not performed, Figure 1 In the mode shown without PCR reaction, the label sequences and the blocking sequences on the detection probe and the universal reporter probe are combined together due to complementary pairing. Therefore, the fluorescent quencher group labeled at the 3' end of the detection probe and the fluorescent reporter group labeled at the 5' end of the universal reporter probe are close to each other, so that the fluorescent reporter group labeled at the 5' end of the universal reporter probe is in a quenched state and cannot emit a fluorescent signal.

[0045] 2. If Figure 1 As shown, when the digital PCR amplification proceeds to the denaturation procedure, the two complementary paired detection probes and the universal reporter probe begin to separate. At this time, if the complementary sequence of the target nucleic acid sequence on the detection probe can be complementary paired with the target nucleic acid sequence of the sample, the upstream primer of the target nucleic acid sequence will extend to the junction of the detection probe and the target nucleic acid sequence, and the nucleic acid polymerase with 5' exonuclease activity and thermal stability in the reaction system will cut the label sequence carried on the detection probe and the blocking sequence marked with a fluorescent quenching group at the 3' end to form two free nucleic acid fragments. Among them, the blocked sequence marked with a fluorescent quenching group at the 3' end can be close to each other through complementary pairing with the complementary sequence of the blocking sequence on the universal reporter probe, and the fluorescent quenching group marked at the 3' end of the blocking sequence will quench the fluorescence of the fluorescent group marked at the 5' end of the universal reporter probe on the complementary pairing side, thereby not generating a fluorescent signal. At the same time, the label sequence in a free state that has been cut off can be complementary paired with the complementary sequence of the label sequence on the universal reporter probe and then subjected to PCR amplification. The amplification proceeds to the area where the closed sequence binds to the 5' end of the universal reporter probe, and the closed sequence is cut, so that the fluorescent group labeled at the 5' end of the universal reporter probe is cut due to the quenching group, thereby releasing the fluorescent signal labeled at the 5' end of the universal reporter probe.

[0046] If the complementary sequence of the target nucleic acid sequence on the detection probe cannot be complementary paired with the target nucleic acid sequence of the sample, when the upstream primer of the target nucleic acid extends to the junction of the detection probe and the target nucleic acid, the nucleic acid polymerase with 5' exonuclease activity and thermal stability in the reaction system cannot cut off the label sequence and the blocking sequence on the detection probe, and therefore cannot form two free nucleic acid fragments, and the subsequent reaction process of complementary pairing and hybridization of the free nucleic acid fragments with the universal reporter probe cannot be carried out, and no corresponding fluorescent signal will be generated.

[0047] The method of the present invention is based on the analysis of the types of fluorescent signals and the intensity of fluorescent signals generated during PCR amplification of universal reporter probes to achieve detection of single-hole multi-target nucleic acid sequences. This process requires the joint action of the label sequence cut from the detection probe, the blocking sequence and the universal reporter probe to generate detectable fluorescent signal types and fluorescent signal intensities.

[0048] Step (2)

[0049] In the method of the present invention, the digital PCR reaction conditions may include: conditions that allow the formation of reaction droplets during the digital PCR reaction, conditions that allow nucleic acid denaturation, conditions that allow nucleic acid annealing and extension, and conditions that allow nucleic acid polymerase to perform an extension reaction.

[0050] In certain embodiments, the digital PCR reaction conditions in step (2) include a nucleic acid polymerase with 5' nuclease activity and other reagents that can be applied to the digital PCR reaction system, wherein the nucleic acid polymerase can use the target nucleic acid sequence as a template to catalyze the extension of the upstream primer and / or can induce the cleavage of the probe. PerfectaMultiplex ToughMix digital PCR premix is ​​particularly preferred, and also includes fluorescein sodium salt for droplet counting.

[0051] In certain embodiments, the digital PCR in step (2) is six-channel digital PCR.

[0052] In certain embodiments, the digital PCR reaction system in step (2) is 6 μL and / or the digital PCR chip is a RUBY chip.

[0053] In certain embodiments, the digital PCR reaction procedure is: droplet generation step: temperature 40°C; pre-denaturation step: temperature 95°C, time 3 minutes; PCR reaction step: denaturation temperature 95°C for 5 seconds, annealing temperature 59°C for 30 seconds, wherein the PCR reaction step is cycled 50 times; pressure release step: temperature 25°C to complete the digital PCR detection process.

[0054] Step (3)

[0055] In the method of the present invention, quantitative detection of the target nucleic acid sequence is achieved by analyzing the type of fluorescent signal and the intensity value of the fluorescent signal after the digital PCR amplification reaction.

[0056] In certain embodiments, the universal reporter probe may include one or two fluorescent groups with different fluorescence value intensities. In this case, in step (3), the analysis of different fluorescence value intensities includes: determining the presence of a certain target nucleic acid sequence based on the different types of fluorescent signals and fluorescent signal intensities obtained.

[0057] Advantageous Effects of the Invention

[0058] Although methods that can be used for multiplex nucleic acid detection have been reported in the prior art, the detection methods cannot meet the needs in many aspects of multiplex nucleic acid detection. The detection method of the present invention adopts different fluorescent signal reporting modes and probe blocking modes. Before the digital PCR reaction, the 3' end of the detection probe is labeled with a fluorescent quenching group, which can be complementary paired with the 5' end of the universal reporter probe labeled with a fluorescent reporter group, so that the universal reporter probe fluorescent group is quenched while maintaining the stability of the universal reporter probe; when the digital PCR reaction is performed, the complementary sequence of the target sequence in the detection probe can be paired with the target sequence DNA of the sample, and the free label sequence and the blocking probe labeled with a fluorescent quenching group at the 3' end are cut out by a nucleic acid polymerase with 5' nuclease activity. The free label sequence can be paired with the complementary sequence of the label sequence on one or more universal reporter probes, and extended under the action of the nucleic acid polymerase until the enzyme cleavage destroys the binding of the fluorescent reporter group and the fluorescent quenching group on the universal reporter probe, so that the fluorescent signal marked on the universal reporter probe produces fluorescence.

[0059] In addition, the method of the present invention is different from the existing disclosed methods in the following aspects: 1. The present invention adopts non-stem-loop fluorescent labeled probes, thereby optimizing the binding efficiency of the probe and the template and improving the intensity of the fluorescent signal; 2. Since the sequences of the six universal reporter probes are fixed, the signal intensity of the fluorescent signal cluster emitted by the universal reporter probe after the amplification reaction is also fixed, thereby eliminating the fluorescence crosstalk phenomenon between different fluorescent signals when the TaqMan probe method performs multiple target nucleic acid detection; 3. The number of detection targets can be increased to 15 by only using universal reporter probes labeled with six different fluorescent groups with fixed sequences; 4. The types and quantities of fluorescent groups modified on each target nucleic acid recognition probe to be detected in the TaqMan probe method are greatly reduced, thereby reducing the detection cost.

[0060] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Shown is a schematic diagram of the detection method of the present invention;

[0062] Figure 2 The blue and Teal dual-channel fluorescence images showing the amplification results of Klebsiella pneumoniae in a single-well single-plex reaction system;

[0063] Figure 3The blue and green dual-channel fluorescence images showing the amplification results of Proteus mirabilis in a single-well single-plex reaction system;

[0064] Figure 4 The blue and yellow dual-channel fluorescence images show the amplification results of Morganella morganii in a single-well single-plex reaction system;

[0065] Figure 5 Shown is a blue and red dual-channel fluorescence image of the amplification result of Salmonella in a single-well single-plex reaction system;

[0066] Figure 6 The display is a dual-channel fluorescence image of Blue and Infra-Red of the amplification result of Enterobacter cloacae in a single-well single-plex reaction system;

[0067] Figure 7 The Teal and Green dual-channel fluorescence images show the amplification results of Staphylococcus aureus in a single-well single-plex reaction system;

[0068] Figure 8 The Teal and Yellow dual-channel fluorescence images show the amplification results of Acinetobacter baumannii in a single-well single-plex reaction system;

[0069] Fig. 9 The Teal and Red dual-channel fluorescence images show the amplification results of Stenotrophomonas maltophilia in a single-well single-plex reaction system;

[0070] Fig.10 Teal and Infra-Red dual-channel fluorescence images showing the amplification results of Streptococcus in a single-well single-plex reaction system;

[0071] Fig.11 Green and Yellow dual-channel fluorescence images showing the amplification results of Enterococcus in a single-well single-plex reaction system;

[0072] Fig.12 Green and Red dual-channel fluorescence images showing the amplification results of Candida species in a single-well single-plex reaction system;

[0073] Fig.13 Green and Infra-Red dual-channel fluorescence images showing the amplification results of Escherichia coli in a single-well single-plex reaction system;

[0074] Fig.14 The yellow and red dual-channel fluorescence images show the amplification results of Pseudomonas aeruginosa in a single-well single-plex reaction system;

[0075] Fig.15 The Yellow and Infra-Red dual-channel fluorescence images show the amplification results of Serratia marcescens in a single-well single-plex reaction system;

[0076] Fig.16 Displays the Red and Infra-Red dual-channel fluorescence images of the internal reference gene amplification results of a single-well single-plex reaction system;

[0077] Fig.17 Shown is a dual-channel fluorescence image of Blue and Teal for the amplification results of Klebsiella pneumoniae in a single-well multiplex reaction system;

[0078] Fig.18 Shown is a blue and green dual-channel fluorescence image of the amplification results of Proteus mirabilis in a single-well multiplex reaction system;

[0079] Fig.19 Shown is a blue and yellow dual-channel fluorescence image of the amplification results of Morganella morganii in a single-well multiplex reaction system;

[0080] Fig. 20 Shown is a blue and red dual-channel fluorescence image of the amplification results of Salmonella in a single-well multiplex reaction system;

[0081] Fig.21 Shown is a dual-channel fluorescence image of Blue and Infra-Red for the amplification results of Enterobacter cloacae in a single-well multiplex reaction system;

[0082] Fig. 22 Shown is the Teal and Green dual-channel fluorescence image of the amplification results of Staphylococcus aureus in a single-well multiplex reaction system;

[0083] Fig.23 Shown is a Teal and Yellow dual-channel fluorescence image of the amplification results of Acinetobacter baumannii in a single-well multiplex reaction system;

[0084] Fig.24 Shown is a Teal and Red dual-channel fluorescence image of the amplification results of Stenotrophomonas maltophilia in a single-well multiplex reaction system;

[0085] Fig.25 Shown is a Teal and Infra-Red dual-channel fluorescence image of the Streptococcus amplification results in a single-well multiplex reaction system;

[0086] Fig.26 Shown is a Green and Yellow dual-channel fluorescence image of the amplification results of Enterococcus in a single-well multiplex reaction system;

[0087] Fig. 27 Shown is a Green and Red dual-channel fluorescence image of the Candida amplification results in a single-well multiplex reaction system;

[0088] Fig.28Shown is the Green and Infra-Red dual-channel fluorescence image of the Escherichia coli amplification results in a single-well multiplex reaction system;

[0089] Fig.29 Shown is the Yellow and Red dual-channel fluorescence image of the amplification results of Pseudomonas aeruginosa in a single-well multiplex reaction system;

[0090] Fig.30 Shown is a dual-channel fluorescence image of Yellow and Infra-Red showing the amplification results of Serratia marcescens in a single-well multiplex reaction system;

[0091] Fig.31 Displays Red and Infra-Red dual-channel fluorescence images of the internal reference gene amplification results in a single-well multiplex reaction system; DETAILED DESCRIPTION

[0092] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0093] The experimental methods for nucleic acid detection involved in the following embodiments are as follows:

[0094] 1. Instruments and reagents

[0095] The primers and probes used in PCR amplification were synthesized by Sangon Biotechnology Co., Ltd.; PerfectaMultiplex ToughMix digital PCR premix was purchased from QUANTABIO; the nucleic acid extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; other conventional chemical reagents were domestic analytical grade reagents.

[0096] 2. Design and synthesis of primers and probes

[0097] 2.1 In the method of the present invention, the fluorescent reporter group is any suitable fluorescent group or molecule known in the art, and specific examples thereof include but are not limited to any one of FAM, HEX, TAMRA, ROX, Cy5, and Cy5.5 labeled with the 5' end.

[0098] 2.2 The optimized universal reporter probe sequence is as follows:

[0099] 5'-FAM-(blocking sequence-tag sequence 1-tag sequence 2-tag sequence 3-tag sequence 4-tag sequence 5) complementary sequence-3';

[0100] 5'-HEX-(blocking sequence-tag sequence 1-tag sequence 6-tag sequence 7-tag sequence 8-tag sequence 9) complementary sequence-3';

[0101] 5'-TAMRA-(blocking sequence-tag sequence 2-tag sequence 6-tag sequence 10-tag sequence 11-tag sequence 12) complementary sequence-3';

[0102] 5'-ROX-(blocking sequence-tag sequence 3-tag sequence 7-tag sequence 10-tag sequence 13-tag sequence 14) complementary sequence-3';

[0103] 5'-Cy5-(blocking sequence-tag sequence 4-tag sequence 8-tag sequence 11-tag sequence 13-tag sequence 15) complementary sequence-3';

[0104] 5'-Cy5.5-(blocking sequence-tag sequence 5-tag sequence 9-tag sequence 12-tag sequence 14-tag sequence 15) complementary sequence-3';

[0105] 2.3 The preferred blocking sequence, tag sequence, complementary sequence of the blocking sequence and tag sequence, and sequence of the universal reporter probe are shown in Table 1 and Table 2, respectively.

[0106] Table 1. Preferred blocking sequences, tag sequences, and complementary sequences of blocking sequences and tag sequences

[0107]

[0108]

[0109] Table 2. Preferred sequences of universal reporter probes

[0110]

[0111] The nucleotide sequences of tag sequence 1 to tag sequence 15 in the universal reporter probe sequence composition shown in Table 2 have common bases or are added with additional 1-2 bases.

[0112] 3. Establishment of single-well single-plex reaction system and reaction procedure

[0113] The digital PCR single-plex reaction system is 6 μL. The reaction system includes 1×Perfecta Multiplex ToughMix, 0.1 μM fluorescein sodium salt, 0.8 μM upstream primer, 0.8 μM downstream primer, 0.4 μM detection probe, 0.4 μM universal reporter probe, and the corresponding volume of template is added according to the concentration. The reaction procedure is: droplet generation step temperature 40°C; pre-denaturation step: temperature 95°C, time 3 minutes; PCR reaction step: denaturation temperature 95°C 5 seconds, annealing temperature 59°C 30 seconds, wherein the PCR reaction step is cycled 40 times; pressure release step: temperature 25°C.

[0114] 4. Establishment of single-well multiple reaction system and reaction procedure

[0115] The digital PCR multiplex reaction system is 6 μL. The reaction system includes 1×Perfecta Multiplex ToughMix, 0.1 μM fluorescein sodium salt, 0.8 μM upstream primer, 0.8 μM downstream primer, 0.4 μM detection probe, 0.4 μM universal reporter probe, and the corresponding volume of template is added according to the concentration. The reaction procedure is: droplet generation step temperature 40°C; pre-denaturation step: temperature 95°C, time 3 minutes; PCR reaction step: denaturation temperature 95°C for 5 seconds, annealing temperature 59°C for 30 seconds, wherein the PCR reaction step is cycled 50 times; pressure release step: temperature 25°C.

[0116] Example 1: Establishment and testing of a single-well single-plex reaction system

[0117] 1. This embodiment uses a single-well detection of a microorganism as the target, and uses 15 wells to detect 14 microorganisms and an internal reference gene. Among them, the names of the 14 microorganisms and the detection primer sequences and probe sequences of the internal reference genes are shown in Table 3, and the types of fluorescent signals that the detected microbial target nucleic acids should have are shown in Table 4.

[0118] Table 3. Microorganism names and their detection primer and probe sequences

[0119]

[0120]

[0121]

[0122] Table 4. Types of fluorescence signals corresponding to 14 microorganisms detected

[0123]

[0124] 2. Determination of single-well single-plex digital PCR detection method

[0125] 2.1 The universal reporter probe with the tag sequence corresponding to the microbial detection probe shown in Table 2 and the primer and detection probe of a microorganism shown in Table 3 are respectively used to detect the single-well single-plex target nucleic acid. The digital PCR reaction system is shown in Table 5. Only one upstream primer, downstream primer, detection probe for detecting the target nucleic acid, the corresponding universal reporter probe and other digital PCR reaction reagents are added to each digital PCR reaction well.

[0126] Table 5. Single-well single-plex digital PCR reaction system

[0127]

[0128] 2.2 Mix the mixture in Table 5 thoroughly and centrifuge it at 1000 rpm for 15 seconds to avoid generating bubbles.

[0129] 2.3 Pipette 5 μL of the reaction solution and load it into the well of the RUBY chip (Catalog#C16011, Stilla Technologies). After the PCR reaction solution is loaded into the RUBY chip, the experiment should be carried out within 30 minutes to avoid affecting the experimental results, and avoid adding bubbles and adding two or more droplets.

[0130] 2.4 Droplet generation and digital PCR amplification

[0131] 2.5 Turn on the Naica Geode droplet generation amplification system and the air pressure pump, and set the digital PCR reaction program as shown in Table 6.

[0132] Table 6. Singleplex digital PCR reaction program

[0133]

[0134] 2.6 Place the RUBY chip on the Naica Geode heating module, confirm that the program is correct, and run the droplet generation and PCR reaction program.

[0135] 2.7 Information Collection

[0136] (1) After the digital PCR reaction is completed, open Naica TM The prism3 droplet reading and analysis system and the "Crystalreader" software ensure that the instrument can be connected and run normally.

[0137] (2) Click the "open tray" button in the "Crystal reader" software interface, place the RUBY chip on the tray, and then click the "close tray" button.

[0138] (3) Click the "New experiment" button in the "Crystal reader" software interface to enter the scanning parameter setting interface: Enter the reaction solution name, chip ID number, sample name and other information in the software setting interface in the order of the digital PCR chip sample well layout table. Strictly check the correspondence between the input information and the chip loading order to ensure that the information is consistent.

[0139] (4) Click the "Scanning Parameters" button in the "Crystal reader" software interface and set the LED exposure time: Blue (65ms), Green (250ms), Red (50ms).

[0140] (5) Click the “SCAN” button in the “Crystal reader” software interface to run the acquisition program.

[0141] 2.8 Result Analysis Method

[0142] (1) Use the "Crystal Miner" software to open the above analysis files respectively. Click the "SETUP" button on the upper right of the software interface, select "Edit Experiment", click the "+" button on the left to load the data file saved in "4. Information Collection", and click "Yes" to confirm the loading.

[0143] (2) Click “ANALYZE DATA” to enter the “2D dot plot” interface of “Plots & Populations”, fine-tune the threshold line according to the droplet distribution, and save the analysis data. It is recommended to use the default threshold line in the “Analysis File”, which can be fine-tuned according to the specific experimental results.

[0144] 3. Analysis of amplification results: Figures 2 to 16 It is shown that after amplification detection, the type and value of fluorescence signals displayed by each microbial target nucleic acid are the same as the types of fluorescence signals shown in Table 4, so the detection results of single-well single-weight of 14 microorganisms are consistent with the expected types of fluorescence signals and can be clearly distinguished.

[0145] Example 2: Establishment and testing of a single-well multiplex digital PCR reaction system

[0146] 1. This example aims to detect 14 microorganisms and an internal reference gene in a single well. Based on the implementation of Example 1, multiple tests and adjustments were carried out to determine the single-well multiplex digital PCR reaction system and reaction procedure. The names of the microorganisms involved and the detection primer and probe sequences are shown in Table 3, and the types of fluorescent signals that the detected microbial target nucleic acids should have are shown in Table 4.

[0147] 2. Determination of single-well multiplex digital PCR detection method

[0148] 2.1 The six universal reporter probes shown in Table 2 and the primers and probes for detecting 14 microorganisms and an internal reference gene shown in Table 3 were used to detect multiple target nucleic acids in a single digital PCR well, that is, 15 upstream and downstream primers, detection probes and six universal reporter probes for detecting target nucleic acids, as well as a certain concentration of template were added to a single digital PCR reaction chip well. The digital PCR reaction system after multiple tests and adjustments is shown in Table 7.

[0149] Table 7. Single-well multiplex digital PCR reaction system

[0150] Component name Use volume (μL) PerfeCTa Multiplex qPCR ToughMix 1.2 Fluorescein sodium salt (2.5 μM) 0.2 Probe mixture (single hole multiple use) 3.6 Template DNA 1 Total volume 6

[0151] 2.2 Mix the mixture in Table 7 thoroughly and centrifuge it at 1000 rpm for 15 seconds to avoid generating bubbles.

[0152] 2.3 Pipette 5 μL of the reaction solution and load it into the well of the RUBY chip (Catalog#C16011, Stilla Technologies). After the PCR reaction solution is loaded into the RUBY chip, the experiment should be carried out within 30 minutes to avoid affecting the experimental results, and avoid adding bubbles and adding two or more droplets.

[0153] 2.4 Droplet generation and digital PCR amplification

[0154] 2.5 Turn on the Naica Geode droplet generation amplification system and the air pressure pump, and set the digital PCR reaction program as shown in Table 8.

[0155] Table 8. Single-well multiplex digital PCR reaction program

[0156]

[0157] 2.6 Place the RUBY chip on the Naica Geode heating module, confirm that the program is correct, and run the droplet generation and PCR reaction program.

[0158] 2.7 Information Collection

[0159] (1) After the digital PCR reaction is completed, open Naica TM The prism3 droplet reading and analysis system and the "Crystalreader" software ensure that the instrument can be connected and run normally.

[0160] (2) Click the "open tray" button in the "Crystal reader" software interface, place the RUBY chip on the tray, and then click the "close tray" button.

[0161] (3) Click the "New experiment" button in the "Crystal reader" software interface to enter the scanning parameter setting interface: Enter the reaction solution name, chip ID number, sample name and other information in the software setting interface in the order of the digital PCR chip sample well layout table. Strictly check the correspondence between the input information and the chip loading order to ensure that the information is consistent.

[0162] (4) Click the "Scanning Parameters" button in the "Crystal reader" software interface and set the LED exposure time: Blue (65ms), Green (250ms), Red (50ms).

[0163] (5) Click the “SCAN” button in the “Crystal reader” software interface to run the acquisition program.

[0164] 2.8 Result Analysis Method

[0165] (1) Use the "Crystal Miner" software to open the above analysis files respectively. Click the "SETUP" button on the upper right of the software interface, select "Edit Experiment", click the left "+" button to load the data file saved in "4. Information Collection", and click "Yes" to confirm the loading.

[0166] (2) Click “ANALYZE DATA” to enter the “2D dot plot” interface of “Plots & Populations”, fine-tune the threshold line according to the droplet distribution, and save the analysis data. It is recommended to use the default threshold line in the “Analysis File”, which can be fine-tuned according to the specific experimental results.

[0167] 3. Analysis of amplification results: Figures 17 to 31 It is shown that after single-well multiplex amplification detection, the type of fluorescence signal displayed by each microbial target nucleic acid is the same as the type of fluorescence signal shown in Table 4, so the detection results of 14 microorganisms in a single digital PCR well are consistent with the expected types of fluorescence signals and can be clearly distinguished.

[0168] The results of the above examples indicate that the method for multiple target nucleic acid detection based on digital PCR described in the present application can be accurately used for digital PCR detection of multiple target nucleic acids in a single well.

[0169] The present application has at least the following advantages: 1. The present invention adopts non-stem-loop fluorescent labeled probes, thereby optimizing the binding efficiency of the probe and the template and improving the intensity of the fluorescent signal; 2. Since the sequences of the six universal reporter probes have been fixed, the signal intensity of the fluorescent signal cluster emitted by the universal reporter probe after the amplification reaction has been fixed, thereby eliminating the fluorescence crosstalk phenomenon between different fluorescent signals when the TaqMan probe method is used for multiple target nucleic acid detection; 3. The number of detection targets can be increased to 15 by only using universal reporter probes labeled with six different fluorescent groups with fixed sequences; 4. The types and quantities of fluorescent groups modified on each target nucleic acid recognition probe to be detected in the TaqMan probe method are greatly reduced, thereby reducing the detection cost.

[0170] It should be understood that although the present application specifically designs primers and probes for the target nucleic acids of 14 microorganisms, those skilled in the art should know that, based on the target nucleic acid primers and probes of the 14 microorganisms in the present application, the target pairing sequences can be adjusted accordingly. At the same time, the design concept based on the detection method of the present application is not limited to the corresponding changes given in the present application.

[0171] The technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, all possible combinations of the technical features in the above-described embodiments are not 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. It should be noted that "in one embodiment", "for example", "for example", etc. in this application are intended to illustrate the present application, rather than to limit the present application. The above-described embodiments only express several implementation methods of the present application, and the description is relatively specific and detailed, but it cannot be understood as a limitation on the scope of the patent application. It should be pointed out that for a person of ordinary skill in the art, without departing from the concept of the present application, several variations and improvements can also be made, which all belong to the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be subject to the attached claims.

Claims

1. A method for detecting multiple target nucleic acids based on digital PCR, comprising the following main steps: (1) Provide the following test components: (a) For each target nucleic acid to be detected, an upstream primer, a downstream primer and a detection probe are provided; the sequence of the detection probe is composed of three parts from 5' to 3', including a tag sequence, a complementary sequence of the partial sequence of the target nucleic acid and a blocking sequence; wherein, The 3' end of the blocking sequence or the interior of the sequence is labeled with one or more fluorescent quenching groups; and when the detection probe hybridizes with the target nucleic acid sequence, the upstream primer is located upstream of the detection probe; (b) providing one or more universal reporter probes for each target nucleic acid to be detected; wherein the sequence of each universal reporter probe is independently composed of two parts from the 5' to the 3' direction, including a complementary sequence to the blocking sequence described in (a) and a complementary sequence to the label sequence; and each universal reporter probe is independently labeled with a different fluorescent group at the 5' end; Wherein, the sequence composition of the universal reporter probe and the fluorescent group labeled at the 5' end are at least one of the following: 5'-FAM-(blocking sequence-tag sequence 1-tag sequence 2-tag sequence 3-tag sequence 4-tag sequence 5) complementary sequence-3'; 5'-HEX-(blocking sequence-tag sequence 1-tag sequence 6-tag sequence 7-tag sequence 8-tag sequence 9) complementary sequence-3'; 5'-TAMRA-(blocking sequence-tag sequence 2-tag sequence 6-tag sequence 10-tag sequence 11-tag sequence 12) complementary sequence-3'; 5'-ROX-(blocking sequence-tag sequence 3-tag sequence 7-tag sequence 10-tag sequence 13-tag sequence 14) complementary sequence-3'; 5'-Cy5-(blocking sequence-tag sequence 4-tag sequence 8-tag sequence 11-tag sequence 13-tag sequence 115) complementary sequence-3'; 5'-Cy5.5-(blocking sequence-tag sequence 5-tag sequence 9-tag sequence 12-tag sequence 14-tag sequence 15) complementary sequence-3'; The blocking sequence, tag sequence and their complementary sequences are shown in the following table: Wherein, the sequence of the universal reporter probe is shown in the following table: (2) preparing a digital PCR reaction system with the sample containing the target nucleic acid to be detected and the components described in (1) and adding the system to a single digital PCR chip well, and running the digital PCR reaction program; (3) After the digital PCR run is completed, the fluorescence type and fluorescence intensity value of the amplified product are analyzed; the analysis of the fluorescence type and fluorescence intensity value includes the fluorescence signal from the fluorescent marker group described in (1)(b), and the corresponding target nucleic acid is determined based on the measured fluorescence signal type of the fluorescent marker group.

2. The method according to claim 1, characterized in that When the detection probe and the universal reporter probe are not subjected to nucleic acid amplification reaction, the label sequence and the blocking sequence on the detection probe and the complementary sequences of the label sequence and the blocking sequence on the universal reporter probe are combined together due to complementary pairing, and the fluorescent quenching group labeled at the 3' end of the detection probe and the fluorescent reporter group labeled at the 5' end of the universal reporter probe are close to each other, so that the fluorescent reporter group labeled at the 5' end of the universal reporter probe is in a quenched state and cannot emit a fluorescent signal.

3. The method according to claim 1, characterized in that One of the detection probes contains a blocking sequence with a fluorescent quenching group at the 3' end, which is cut during the nucleic acid amplification reaction. The blocking sequence with a fluorescent quenching group at the 3' end after cutting can be complementary paired with the complementary sequence of the blocking sequence on the universal reporter probe. The fluorescent quenching group labeled at the 3' end of the blocking sequence quenches the fluorescence of the fluorescent group labeled at the 5' end of the complementary paired universal reporter probe.

4. The method according to claim 1, characterized in that: One of the detection probes contains a label sequence that is cleaved during a nucleic acid amplification reaction. The 5' end of the cleaved label sequence is complementary paired with a sequence on the universal reporter probe that is complementary to the label sequence and then PCR amplified. When the 3' end of the universal reporter probe that has been amplified to the 5' end carries a fluorescent quenching group, the fluorescent quenching group on the 3' end of the blocking sequence is cleaved, thereby causing the fluorescent group labeled on the 5' end of the universal reporter probe to produce a fluorescent signal.

5. The method according to claim 1, characterized in that The digital PCR reaction system in step (2) of the method includes using a digital PCR premix and fluorescein sodium salt, wherein the digital PCR premix includes dNTPs, a nucleic acid polymerase having 5' exonuclease activity and thermal stability, and a PCR buffer.

6. The method according to claim 1, characterized in that The digital PCR is a six-channel digital PCR.

7. The method according to claim 5, characterized in that In step (2) of the method, the digital PCR reaction system is 6 μL and / or, the PCR chip is a RUBY chip, and the corresponding PCR reaction procedures are as follows: Droplet generation step: temperature 40°C; Pre-denaturation step: temperature 95°C, time 3 minutes; PCR reaction step: denaturation temperature 95°C for 5 seconds, annealing temperature 59°C for 30 seconds, wherein the PCR reaction step is cycled 50 times; Pressure release step: temperature 25°C.

Images