Digital PCR-based method for detecting multiple target nucleic acids

Abstract

Provided is a six-channel digital PCR-based method using a non-stem-loop universal reporter probe and a detection probe in which primers for multiple target nucleic acids, the detection probe, the universal reporter probe, and other reagents are added to a single reaction well for amplification and special analysis. In the method, the use of just a fixed-sequence universal reporter probe labeled with six different fluorescent groups in a single well can increase the number of detected targets to 15, eliminating the fluorescence crosstalk which occurs between different fluorescent signals when the TaqMan probe method is used to detect multiple target nucleic acids, and significantly reducing the variety and quantity of modification fluorescent groups on each probe that recognizes a target nucleic acid to be detected in the TaqMan probe method, thereby reducing the detection costs.

Description

A method for multiplex target nucleic acid detection based on digital PCR Technical Field

[0001] This application relates to the multiplex detection of nucleic acid molecules. Specifically, it provides a method for multiplex target nucleic acid detection based on digital PCR, which enables the quantitative detection of multiple target nucleic acid sequences in a single-well reaction system. Background Technology

[0002] In recent years, multiplex quantitative PCR has been developed, which integrates the detection of multiple target nucleic acids into a single reaction tube. This method saves precious samples, reduces detection costs, and retains the accuracy and sensitivity of quantitative PCR. However, the maximum number of target nucleic acid sequences that this method can detect depends on the number of channels in the quantitative PCR instrument and the influence of crosstalk between fluorescence signals. Therefore, the number of targets that can be detected in a single reaction tube is still relatively small. Digital PCR is an absolute quantitative nucleic acid detection method, and there are currently attempts to detect multiple target nucleic acids in a single reaction well. However, in applications using 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 are also limited by the number of fluorescence detection channels in the instrument and the influence of crosstalk between fluorescence signals. Therefore, it is very important to develop a new digital PCR method that is different from the TaqMan probe method for detecting multiple target nucleic acids in a single reaction well. This method can not only save samples and reduce costs, but also accurately quantify multiple target nucleic acids. Summary of the Invention

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

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

[0005] (1) Provide the following detection 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 consists of three parts from the 5' to 3' direction, including a tag sequence, a complementary sequence to a portion of the target nucleic acid sequence, and a blocking sequence; wherein, the 3' end or the interior of the blocking 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) For each target nucleic acid to be detected, one or more universal reporter probes are provided; wherein each universal probe is independently composed of two parts from the 5' to 3' direction, including the complementary sequence of the closed sequence described in (a) and the complementary sequence of the tag sequence; and each universal reporter probe is independently labeled with a different fluorescent group at the 5' end;

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

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

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

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

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

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

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

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

[0016]

[0017] The sequence of the universal reporting probe is shown in the table below:

[0018]

[0019] In the optimized universal reporter probe sequence composition shown in the table above, the nucleotide sequences of tag sequences 1 to 15 have shared bases or have 1-2 additional bases.

[0020] (2) Prepare a digital PCR reaction system with the sample containing the target nucleic acid sequence and contact it with the components described in (1) in a single digital PCR chip well, and run the digital PCR reaction program;

[0021] (3) After the digital PCR run is completed, the fluorescence type and fluorescence intensity value of the amplification product are analyzed; the analysis of fluorescence type and fluorescence intensity value includes the fluorescence signal from the fluorescent labeling group described in (1)(b), and the corresponding target nucleic acid sequence is determined based on the fluorescence signal type of the fluorescent labeling group.

[0022] In some implementations, for a six-channel digital PCR detection method, the number of detection probes can be at least one, at least two, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0023] In some implementations, for a six-channel digital PCR detection method, the number of universal reporter probes can be 1, 2, 3, 4, 5, or 6.

[0024] Step (1)

[0025] Detection probe

[0026] As shown in Figure 1, the detection probe sequence consists of three parts from 5' to 3': a tag sequence, a complementary sequence to the target nucleic acid portion, and a blocking sequence. The 3' end of the blocking sequence is labeled with one or more, but only, fluorescent quenching groups. Therefore, the detection probe is also labeled only with fluorescent quenching groups and not with fluorescent reporter groups. In some embodiments, the fluorescent quenching groups labeled at the 3' end or inside the blocking sequence of the detection probe are molecules or groups capable of absorbing / quenching the fluorescence, such as BHQ-1 or BHQ-2.

[0027] General Reporting Probe

[0028] As shown in Figure 1, the sequence of the universal reporter probe consists of two parts from the 5' to 3' direction: the complementary sequence of the blocking sequence and the complementary sequence of the tag sequence. The 5' end of the universal reporter probe is labeled with a fluorescent reporter group, which can be any suitable group or molecule known in the art. Specific examples include, but are not limited to, any one of FAM, HEX, TAMRA, ROX, Cy5, and Cy5.5 as the fluorescent group labeled at the 5' end of the universal reporter probe described in step (1) (b).

[0029] The optimized universal report probe sequence is as follows:

[0030] 5'-FAM-(closed sequence-tag sequence 1-tag sequence 2-tag sequence 3-tag sequence 4-tag sequence 5) complementary sequence-3';

[0031] 5'-HEX-(closed sequence-tag sequence 1-tag sequence 6-tag sequence 7-tag sequence 8-tag sequence 9) complementary sequence-3';

[0032] 5'-TAMRA-complementary sequence of (closed sequence-tag sequence 2-tag sequence 6-tag sequence 10-tag sequence 11-tag sequence 12)-3';

[0033] 5'-ROX-complementary sequence of (closed sequence-tag sequence 3-tag sequence 7-tag sequence 10-tag sequence 13-tag sequence 14)-3';

[0034] 5'-Cy5-complementary sequence of (closed sequence-tag sequence 4-tag sequence 8-tag sequence 11-tag sequence 13-tag sequence 15)-3';

[0035] 5'-Cy5.5-(closed sequence-tag sequence 5-tag sequence 9-tag sequence 12-tag sequence 14-tag sequence 15) complementary sequence-3';

[0036] The closed sequence, the tag sequence, and the complementary sequence of the tag sequence are shown in the table below:

[0037]

[0038] The sequence composition of the general report probe is shown in the table below:

[0039]

[0040] In the optimized universal reporter probes listed in the table above, the nucleotide sequences of tag sequences 1 to 15 share common bases or have 1-2 additional bases added.

[0041] How do detection probes and universal reporting probes work?

[0042] 1. When no PCR amplification reaction is performed, as shown in Figure 1, the tag sequences and blocking sequences on the detection probe and the universal reporter probe are paired together due to their 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 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.

[0043] 2. As shown in Figure 1, when digital PCR amplification reaches the denaturation stage, the two complementary detection probes and the universal reporter probe begin to separate. If the complementary sequence of the target nucleic acid sequence on the detection probe can hybridize with the target nucleic acid sequence of the sample, the upstream primer of the target nucleic acid sequence extends to the binding site between the detection probe and the target nucleic acid sequence. The nucleic acid polymerase in the reaction system, possessing 5' exonuclease activity and thermostability, will cleave the tag sequence carried on the detection probe and the blocking sequence labeled with a fluorescence quencher at the 3' end, forming two free nucleic acid fragments. The cleaved blocking sequence labeled with a fluorescence quencher at the 3' end can then pair with the complementary sequence of the blocking sequence on the universal reporter probe, causing them to approach each other. The fluorescence quencher at the 3' end of the blocking sequence will then quench the fluorescence of the fluorescent group labeled with the 5' end of the universal reporter probe on the complementary pairing side, thus preventing the generation of a fluorescence signal. Meanwhile, the cleaved, free tag sequence can pair with the complementary sequence of the tag sequence on the universal reporter probe for PCR amplification. When the amplification reaches the region where the blocking sequence binds to the 5' end of the universal reporter probe, the blocking sequence is cleaved, thereby releasing the fluorescent signal labeled at the 5' end of the universal reporter probe due to the cleavage of the quenching group.

[0044] If the complementary sequence of the target nucleic acid sequence on the detection probe cannot hybridize with the target nucleic acid sequence of the sample, then when the upstream primer of the target nucleic acid extends to the binding site between the detection probe and the target nucleic acid, the nucleic acid polymerase with 5' exonuclease activity and thermostability in the reaction system cannot cut off the tag sequence and blocking sequence on the detection probe. Therefore, two free nucleic acid fragments cannot be formed, and the subsequent reaction process of complementary hybridization between the free nucleic acid fragments and the universal reporter probe cannot proceed, and no corresponding fluorescent signal will be generated.

[0045] The method of this invention is based on the analysis of the types and intensities of fluorescence signals generated during PCR amplification of a universal reporter probe to achieve the detection of multi-target nucleic acid sequences in a single well. This process requires the combined action of the tag sequence cut from the detection probe, the blocking sequence, and the universal reporter probe to generate detectable types and intensities of fluorescence signals.

[0046] Step (2)

[0047] 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 extension, and conditions that allow nucleic acid polymerase to perform extension reactions.

[0048] In some embodiments, the digital PCR reaction conditions in step (2) include a nucleic acid polymerase with 5' nuclease activity suitable for digital PCR reaction systems, as well as other reagents, wherein the nucleic acid polymerase is capable of catalyzing the extension of the upstream primer and / or inducing probe cleavage using the target nucleic acid sequence as a template. Perfecta Multiplex ToughMix digital PCR premix is ​​particularly preferred, and it also includes sodium fluorescein for droplet counting.

[0049] In some implementations, the digital PCR in step (2) is a six-channel digital PCR.

[0050] In some implementations, the digital PCR reaction system in step (2) is 6 μL and / or the digital PCR chip is a RUBY chip.

[0051] In some implementations, the digital PCR reaction procedure is 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 this PCR reaction step is cycled 50 times; decompression step: temperature 25°C to complete the digital PCR detection process.

[0052] Step (3)

[0053] In the method of the present invention, quantitative detection of target nucleic acid sequences is achieved by analyzing the types and intensity values ​​of fluorescence signals after digital PCR amplification reaction.

[0054] In some implementations, the universal reporter probe may contain one or two fluorescent groups with different fluorescence intensities. In this case, the analysis of different fluorescence intensities in step (3) includes determining the presence of a target nucleic acid sequence based on the different types and intensities of the obtained fluorescence signals. Beneficial effects of the invention

[0055] While existing technologies have reported methods for multiplex nucleic acid detection, they often fall short in many aspects. This invention employs different fluorescent signal reporting modes and probe blocking modes. Before digital PCR, the 3' end of the detection probe is labeled with a fluorescent quencher group, which can complementaryly pair with the 5' end of a universal reporter probe labeled with a fluorescent reporter group. This allows the universal reporter probe to maintain its stability while quenching the fluorescent group. During digital PCR, the complementary sequence of the target sequence in the detection probe pairs with the target DNA sequence in the sample. A 5' nuclease polymerase then cleaves the free tag sequence and the blocking probe labeled with the fluorescent quencher group at the 3' end. The free tag sequence can pair with the complementary sequence of the tag sequence on one or more universal reporter probes and extend under the action of the nucleic acid polymerase until the enzyme cleaves and disrupts the binding between the fluorescent reporter group and the fluorescent quencher group on the universal reporter probe, thus generating fluorescence on the universal reporter probe.

[0056] In addition, the method in this invention differs from existing methods in the following aspects: 1. This invention uses a non-stem-loop fluorescently labeled probe, thereby optimizing the binding efficiency between the probe and the template and improving the intensity of the fluorescence signal; 2. Since the sequences of the six universal reporter probes are fixed, the signal intensity of the fluorescence signal cluster emitted by the universal reporter probe after the amplification reaction is also fixed, thereby eliminating fluorescence crosstalk between different fluorescence signals when performing multiple target nucleic acid detection in the TaqMan probe method; 3. The number of detection targets can be increased to 15 by using only six universal reporter probes labeled with six different fluorescent groups with fixed sequences; 4. The types and number of fluorescent groups modified on each target nucleic acid recognition probe in the TaqMan probe method are significantly reduced, thus reducing detection costs.

[0057] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0058] Figure 1 shows a schematic diagram of the detection method of the present invention;

[0059] Figure 2 shows the Blue and Teal dual-channel fluorescence images of the Klebsiella pneumoniae amplification results in a single-well single-reaction system;

[0060] Figure 3 shows the Blue and Green dual-channel fluorescence images of the amplification results of Proteus mirabilis in a single-well single-reaction system;

[0061] Figure 4 shows the Blue and Yellow dual-channel fluorescence spectrum of the amplification results of Morganella morganii in a single-well single-reaction system.

[0062] Figure 5 shows the Blue and Red dual-channel fluorescence images of the Salmonella amplification results in a single-well single-reaction system;

[0063] Figure 6 shows the Blue and Infra-Red dual-channel fluorescence images of the amplification results of Enterobacter cloacae in a single-well single-reaction system;

[0064] Figure 7 shows the Teal and Green dual-channel fluorescence images of the Staphylococcus aureus amplification results in a single-well single-reaction system;

[0065] Figure 8 shows the Teal and Yellow dual-channel fluorescence spectra of Acinetobacter baumannii amplification results in a single-well single-reaction system;

[0066] Figure 9 shows the Teal and Red dual-channel fluorescence spectra of the amplification results of Stenotrophomonas maltophilia in a single-well single-reaction system;

[0067] Figure 10 shows the Teal and Infra-Red dual-channel fluorescence images of the amplification results of Streptococcus spp. in a single-well single-reaction system;

[0068] Figure 11 shows the Green and Yellow dual-channel fluorescence images of the amplification results of Enterococcus spp. in a single-well single-reaction system;

[0069] Figure 12 shows the Green and Red dual-channel fluorescence images of the Candida amplification results in a single-well single-reaction system.

[0070] Figure 13 shows the Green and Infra-Red dual-channel fluorescence images of the Escherichia coli amplification results in a single-well single-reaction system.

[0071] Figure 14 shows the Yellow and Red dual-channel fluorescence images of the amplification results of Pseudomonas aeruginosa in a single-well single-reaction system.

[0072] Figure 15 shows the Yellow and Infra-Red dual-channel fluorescence images of the amplification results of Serratia marcescens in a single-well single-reaction system;

[0073] Figure 16 shows the Red and Infra-Red dual-channel fluorescence spectra of the amplification results of the internal reference gene in a single-well single-reaction system;

[0074] Figure 17 shows the Blue and Teal dual-channel fluorescence images of the Klebsiella pneumoniae amplification results in a single-well multiplex reaction system;

[0075] Figure 18 shows the Blue and Green dual-channel fluorescence images of the amplification results of Proteus mirabilis in a single-well multiplex reaction system;

[0076] Figure 19 shows the Blue and Yellow dual-channel fluorescence spectrum of the amplification results of Morganella morganii in a single-well multiplex reaction system.

[0077] Figure 20 shows the Blue and Red dual-channel fluorescence images of Salmonella amplification results in a single-well multiplex reaction system;

[0078] Figure 21 shows the Blue and Infra-Red dual-channel fluorescence images of the amplification results of Enterobacter cloacae in a single-well multiplex reaction system;

[0079] Figure 22 shows the Teal and Green dual-channel fluorescence images of Staphylococcus aureus amplification results in a single-well multiplex reaction system;

[0080] Figure 23 shows the Teal and Yellow dual-channel fluorescence spectra of Acinetobacter baumannii amplification results in a single-well multiplex reaction system;

[0081] Figure 24 shows the Teal and Red dual-channel fluorescence images of the amplification results of Stenotrophomonas maltophilia in a single-well multiplex reaction system;

[0082] Figure 25 shows the Teal and Infra-Red dual-channel fluorescence images of the amplification results of Streptococcus spp. in a single-well multiplex reaction system;

[0083] Figure 26 shows the Green and Yellow dual-channel fluorescence images of the amplification results of Enterococcus spp. in a single-well multiplex reaction system;

[0084] Figure 27 shows the Green and Red dual-channel fluorescence images of the Candida amplification results in a single-well multiplex reaction system;

[0085] Figure 28 shows the Green and Infra-Red dual-channel fluorescence images of the amplification results of Escherichia coli in a single-well multiplex reaction system;

[0086] Figure 29 shows the Yellow and Red dual-channel fluorescence images of the amplification results of Pseudomonas aeruginosa in a single-well multiplex reaction system;

[0087] Figure 30 shows the Yellow and Infra-Red dual-channel fluorescence images of the amplification results of Serratia marcescens in a single-well multiplex reaction system;

[0088] Figure 31 shows the Red and Infra-Red dual-channel fluorescence spectra of the amplification results of the internal reference gene in a single-well multiplex reaction system. Detailed Implementation

[0089] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0090] The experimental methods for nucleic acid detection involved in the following examples are described below:

[0091] 1. Instruments and reagents

[0092] Primers and probes used for PCR amplification were synthesized by Sangon Biotech Co., Ltd.; Perfecta Multiplex ToughMix digital PCR premix was purchased from QUANTABIO; nucleic acid extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; other conventional chemical reagents were domestically produced analytical grade reagents.

[0093] 2. Design and synthesis of primers and probes

[0094] 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 include, but are not limited to, any one of FAM, HEX, TAMRA, ROX, Cy5, and Cy5.5 labeled at the 5' end.

[0095] 2.2 The optimized universal report probe sequence consists of the following:

[0096] 5'-FAM-(closed sequence-tag sequence 1-tag sequence 2-tag sequence 3-tag sequence 4-tag sequence 5) complementary sequence-3';

[0097] 5'-HEX-(closed sequence-tag sequence 1-tag sequence 6-tag sequence 7-tag sequence 8-tag sequence 9) complementary sequence-3';

[0098] 5'-TAMRA-complementary sequence of (closed sequence-tag sequence 2-tag sequence 6-tag sequence 10-tag sequence 11-tag sequence 12)-3';

[0099] 5'-ROX-complementary sequence of (closed sequence-tag sequence 3-tag sequence 7-tag sequence 10-tag sequence 13-tag sequence 14)-3';

[0100] 5'-Cy5-complementary sequence of (closed sequence-tag sequence 4-tag sequence 8-tag sequence 11-tag sequence 13-tag sequence 15)-3';

[0101] 5'-Cy5.5-(closed sequence-tag sequence 5-tag sequence 9-tag sequence 12-tag sequence 14-tag sequence 15) complementary sequence-3';

[0102] 2.3 The preferred closed sequence, tag sequence, complementary sequence of closed sequence and tag sequence, and universal reporter probe sequence are shown in Table 1 and Table 2, respectively.

[0103] Table 1. Preferred closed sequences, tag sequences, and complementary sequences of closed and tag sequences.

[0104]

[0105] Table 2. Preferred sequences of universal reporter probes

[0106]

[0107] In the universal reporter probe sequence composition shown in Table 2, the nucleotide sequences of tag sequences 1 to 15 have shared bases or have 1-2 additional bases.

[0108] 3. Establishment of a single-hole single-response system and reaction procedure

[0109] The digital PCR singleton reaction system consisted of 6 μL. The reaction system included 1×Perfecta Multiplex ToughMix, 0.1 μM sodium fluorescein, 0.8 μM upstream primer, 0.8 μM downstream primer, 0.4 μM detection probe, and 0.4 μM universal reporter probe, with the appropriate volume of template added according to the concentration. The reaction program was as follows: droplet generation step at 40℃; pre-denaturation step at 95℃ for 3 minutes; PCR reaction step: denaturation at 95℃ for 5 seconds, annealing at 59℃ for 30 seconds, with this PCR reaction step repeated 40 times; decompression step at 25℃.

[0110] 4. Establishment of single-well multiple reaction system and reaction procedure

[0111] The digital PCR multiplex reaction system consisted of 6 μL. The reaction system included 1×Perfecta Multiplex ToughMix, 0.1 μM sodium fluorescein, 0.8 μM upstream primer, 0.8 μM downstream primer, 0.4 μM detection probe, and 0.4 μM universal reporter probe, with the appropriate volume of template added according to the concentration. The reaction program was as follows: droplet generation step at 40℃; pre-denaturation step at 95℃ for 3 minutes; PCR reaction step: denaturation at 95℃ for 5 seconds, annealing at 59℃ for 30 seconds, with this PCR reaction step repeated 50 times; decompression step at 25℃.

[0112] Example 1: Establishment and testing of a single-pore single-reaction system

[0113] 1. In this embodiment, the detection of one microorganism per well is used as the target, and 14 microorganisms and one internal reference gene are detected using 15 wells. The names of the 14 microorganisms and the detection primer and probe sequences of the internal reference gene are shown in Table 3, and the types of fluorescent signals that the target nucleic acid of the detected microorganisms should have are shown in Table 4.

[0114] Table 3. Names of Microorganisms and Their Detection Primers and Probe Sequences

[0115]

[0116] Table 4. Types of fluorescence signals after detection of 14 microorganisms

[0117]

[0118] 2. Determination of the single-well single-pair digital PCR detection method

[0119] 2.1 The universal reporter probes with tag sequences corresponding to the microbial detection probes shown in Table 2, and the primers and detection probes for one type of microorganism shown in Table 3, were used for single-well single-target nucleic acid detection. The digital PCR reaction system is shown in Table 5. Only one upstream primer, downstream primer, detection probe, corresponding universal reporter probe, and other digital PCR reaction reagents for detecting the target nucleic acid were added to each digital PCR reaction well.

[0120] Table 5. Single-well single-particle digital PCR reaction system

[0121]

[0122] 2.2 Thoroughly mix the mixture in Table 5 and centrifuge at 1000 rpm for 15 seconds to avoid generating bubbles.

[0123] 2.3 Transfer 5 μL of reaction solution into the wells of the RUBY chip (Catalog#C16011, Stilla Technologies). After loading the PCR reaction solution into the RUBY chip, perform the experiment within 30 minutes to avoid affecting the experimental results. At the same time, avoid adding air bubbles and avoid adding two or more droplets.

[0124] 2.4 Droplet generation and digital PCR amplification

[0125] 2.5 Turn on the Naica Geode droplet generation amplification system and the pressure pump, and set the digital PCR reaction program as shown in Table 6.

[0126] Table 6. Singleton Digital PCR Reaction Procedure

[0127]

[0128] 2.6 Place the RUBY chip on the Naica Geode heating module. After confirming that the program is correct, run the droplet generation and PCR reaction programs.

[0129] 2.7 Information Collection

[0130] (1) After the digital PCR reaction is complete, open Naica TM The Prism3 droplet reading and analysis system and the "Crystal Reader" software ensure that the instrument can be properly linked and operated.

[0131] (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.

[0132] (3) Click the "New experiment" button in the "Crystal reader" software interface to enter the scanning parameter setting interface: According to the order of the digital PCR chip sample well layout table, enter the reaction solution name, Chip ID number, sample name, etc. in the software setting interface. Strictly check the correspondence between the input information and the chip sample loading order to ensure that the information is consistent.

[0133] (4) Click the “Scanning Parameters” button in the “Crystal reader” software interface and set the LED exposure time: Blue (65 ms), Green (250 ms), Red (50 ms).

[0134] (5) Click the “SCAN” button in the “Crystal reader” software interface to run the acquisition program.

[0135] 2.8 Result Analysis Methods

[0136] (1) Use the "Crystal Miner" software to open the above analysis files respectively, click the "SETUP" button in the upper right corner of the software interface, select "Edit Experiment", click the "+" button on the left, load the data file saved in "4. Information Collection", and click "Yes" to confirm loading.

[0137] (2) Click “ANALYZE DATA” to enter the “Plots & Populations” “2D dot plot” interface, 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.

[0138] 3. Analysis of amplification results: As shown in Figures 2 to 16, after amplification and detection, the types and values ​​of fluorescence signals displayed by each microbial target nucleic acid are the same as those shown in Table 4. Therefore, the detection results of 14 microorganisms in single wells are consistent with the expected types of fluorescence signals and can be clearly distinguished.

[0139] Example 2: Establishment and testing of a single-well multiplex digital PCR reaction system

[0140] 1. This embodiment aims to detect 14 microorganisms and one internal reference gene in a single well. Based on the implementation of Example 1, multiple tests and adjustments were performed to determine the single-well multiplex digital PCR reaction system and reaction procedure. The names of the microorganisms involved, as well as the detection primers and probe sequences, are shown in Table 3. The types of fluorescent signals that the target nucleic acids of the microorganisms should have are shown in Table 4.

[0141] 2. Determination of Single-Well Multiplex Digital PCR Detection Method

[0142] 2.1 The six universal reporter probes shown in Table 2 and the primers and probes shown in Table 3 for detecting 14 microorganisms and one internal reference gene were used to perform multiplex target nucleic acid detection in a single well of a digital PCR chip. That is, 15 upstream and downstream primers for detecting target nucleic acids, detection probes and six universal reporter probes, as well as a certain concentration of template were added to a single well of a digital PCR reaction chip. The digital PCR reaction system after multiple tests and adjustments is shown in Table 7.

[0143] Table 7. Single-well multiplex digital PCR reaction system

[0144]

[0145] 2.2 Thoroughly mix the mixture in Table 7 and centrifuge at 1000 rpm for 15 seconds to avoid generating bubbles.

[0146] 2.3 Transfer 5 μL of reaction solution into the wells of the RUBY chip (Catalog#C16011, Stilla Technologies). After loading the PCR reaction solution into the RUBY chip, perform the experiment within 30 minutes to avoid affecting the experimental results. At the same time, avoid adding air bubbles and avoid adding two or more droplets.

[0147] 2.4 Droplet generation and digital PCR amplification

[0148] 2.5 Turn on the Naica Geode droplet generation amplification system and the pressure pump, and set the digital PCR reaction program as shown in Table 8.

[0149] Table 8. Single-well multiplex digital PCR reaction procedure

[0150]

[0151] 2.6 Place the RUBY chip on the Naica Geode heating module. After confirming that the program is correct, run the droplet generation and PCR reaction programs.

[0152] 2.7 Information Collection

[0153] (1) After the digital PCR reaction is complete, open Naica TM The Prism3 droplet reading and analysis system and the "Crystal Reader" software ensure that the instrument can be properly linked and operated.

[0154] (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.

[0155] (3) Click the "New experiment" button in the "Crystal reader" software interface to enter the scanning parameter setting interface: According to the order of the digital PCR chip sample well layout table, enter the reaction solution name, Chip ID number, sample name, etc. in the software setting interface. Strictly check the correspondence between the input information and the chip sample loading order to ensure that the information is consistent.

[0156] (4) Click the “Scanning Parameters” button in the “Crystal reader” software interface and set the LED exposure time: Blue (65 ms), Green (250 ms), Red (50 ms).

[0157] (5) Click the “SCAN” button in the “Crystal reader” software interface to run the acquisition program.

[0158] 2.8 Result Analysis Methods

[0159] (1) Use the “Crystal Miner” software to open the above analysis files respectively, click the “SETUP” button in the upper right corner of the software interface, select “Edit Experiment”, click the left “+” button, load the data file saved in “4. Information Collection”, and click “Yes” to confirm loading.

[0160] (2) Click “ANALYZE DATA” to enter the “Plots & Populations” “2D dot plot” interface, 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.

[0161] 3. Analysis of amplification results: Figures 17 to 31 show that after single-well multiplex amplification detection, the types of fluorescence signals displayed by each microbial target nucleic acid are the same as those shown in Table 4. Therefore, the detection results of 14 microorganisms in a single well of digital PCR are consistent with the expected types of fluorescence signals and can be clearly distinguished.

[0162] The results of the above embodiments demonstrate that the method for multiplex target nucleic acid detection based on digital PCR described in this application can be accurately used for single-well multiplex target nucleic acid digital PCR detection.

[0163] This application has at least the following advantages: 1. The present invention uses a non-stem-loop fluorescently labeled probe, thereby optimizing the binding efficiency between the probe and the template and improving the intensity of the fluorescence signal; 2. Since the sequences of the six universal reporter probes are fixed, the signal intensity of the fluorescent signal clusters emitted by the universal reporter probes after the amplification reaction is fixed, thereby eliminating the fluorescence crosstalk phenomenon between different fluorescence signals when performing multiple target nucleic acid detection in the TaqMan probe method; 3. The number of detection targets can be increased to 15 by using only six universal reporter probes labeled with six different fluorescent groups with fixed sequences; 4. The types and number of fluorescent groups modified on each target nucleic acid recognition probe in the TaqMan probe method are greatly reduced, thus reducing the detection cost.

[0164] It should be understood that although this application specifically designs primers and probes for the target nucleic acids of 14 microorganisms, those skilled in the art should know that the target pairing sequences can be adjusted accordingly based on the target nucleic acid primers and probes for the 14 microorganisms in this application. Furthermore, the design concept based on the detection method in this application is not limited to the corresponding variations given in this application.

[0165] The technical features of the embodiments described above can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. It should be noted that the terms "in one embodiment," "for example," and "again," etc., in this application are intended to illustrate the application and not to limit it. The embodiments described above only illustrate several implementation methods of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent application. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for detecting multiplex target nucleic acids based on digital PCR, comprising the following main steps: (1) Provide the following detection components: (a) for each target nucleic acid to be detected, providing an upstream primer, a downstream primer and a detection probe; the sequence of the detection probe consists of three parts from 5' to 3' direction, which comprises a tag sequence, a complementary sequence of a part of the target nucleic acid and a blocking sequence; wherein, The 3' end of the closed 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) For each target nucleic acid to be detected, one or more universal reporter probes are provided; wherein the sequence of each universal reporter probe is independently composed of two parts from the 5' to 3' direction, including a complementary sequence to the closed sequence described in (a) and a complementary sequence to the tag sequence; and each universal reporter probe is independently labeled with a different fluorescent group at the 5' end; The sequence composition of the universal reporter probe and the fluorescent group labeled at its 5' end are at least one of the following: 5'-FAM-(closed sequence-tag sequence 1-tag sequence 2-tag sequence 3-tag sequence 4-tag sequence 5) complementary sequence-3'; 5'-HEX-(closed sequence-tag sequence 1-tag sequence 6-tag sequence 7-tag sequence 8-tag sequence 9) complementary sequence-3'; 5'-TAMRA-complementary sequence of (closed sequence-tag sequence 2-tag sequence 6-tag sequence 10-tag sequence 11-tag sequence 12)-3'; 5'-ROX-complementary sequence of (closed sequence-tag sequence 3-tag sequence 7-tag sequence 10-tag sequence 13-tag sequence 14)-3'; 5'-Cy5-complementary sequence of (closed sequence-tag sequence 4-tag sequence 8-tag sequence 11-tag sequence 13-tag sequence 115)-3'; 5'-Cy5.5-(closed sequence-tag sequence 5-tag sequence 9-tag sequence 12-tag sequence 14-tag sequence 15) complementary sequence-3'; The closed sequence, the tag sequence, and their complementary sequences are shown in the table below: The sequence of the universal reporting probe is shown in the table below: (2) Prepare a digital PCR reaction system by combining the sample containing the target nucleic acid with the components described in (1) and add it into a single digital PCR chip well, and run the digital PCR reaction program; (3) After the digital PCR run is completed, the fluorescence type and fluorescence intensity value of the amplification product are analyzed; the analysis of fluorescence type and fluorescence intensity value includes the fluorescence signal from the fluorescent labeling group described in (1) (b), and the corresponding target nucleic acid is determined based on the fluorescence signal type of the fluorescent labeling group.

2. The method of claim 1, wherein, When no nucleic acid amplification reaction is performed, the complementary sequences of the tag sequence and blocking sequence on the detection probe and the tag sequence and blocking sequence on the universal reporter probe are bound together due to complementary pairing. 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.

3. The method of claim 1, wherein, One of the detection probes comprises a blocking sequence with a fluorescent quenching group at its 3' end, which is cleaved during nucleic acid amplification. The cleaved blocking sequence with a fluorescent quenching group at its 3' end can be complementary to 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 universal reporter probe.

4. The method of claim 1, wherein, One of the detection probes includes a tag sequence that is cleaved during nucleic acid amplification. The 5' end of the cleaved tag sequence is complementary to a sequence on the universal reporter probe that is complementary to the tag sequence, and then PCR amplification is performed. When the 3' end of the universal reporter probe, which has been bound to the 5' end, has a fluorescent quencher group, the fluorescent quencher 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 generate a fluorescent signal.

5. The method of claim 1, wherein, The digital PCR reaction system in step (2) of the method includes the use of digital PCR premix and sodium fluorescein. The digital PCR premix includes dNTPs, a nucleic acid polymerase with 5' exonuclease activity and thermostability, and PCR buffer.

6. The method of claim 1, wherein, The digital PCR described is a six-channel digital PCR.

7. The method of claim 5, wherein, 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 program is as follows: Droplet formation steps: Temperature 40℃; Pre-denaturation step: temperature 95℃, time 3 minutes; PCR reaction steps: denaturation temperature 95℃ for 5 seconds, annealing temperature 59℃ for 30 seconds, and this PCR reaction step is repeated 50 times. Decompression procedure: Temperature 25℃.

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