A nucleic acid complex and a multiplex nucleic acid detection method

Abstract

The application discloses a nucleic acid complex and a multiple nucleic acid detection method, and belongs to the technical field of fluorescence quantitative PCR detection. The ternary nucleic acid complex is composed of first, second and third nucleic acids. The first nucleic acid is modified with a fluorescent group and a quencher group. The second nucleic acid is modified with a fluorescent group and a quencher group and is 3' phosphate modified. The third nucleic acid is only modified with a quencher group at the 5' end. The second and third nucleic acids are complementary to each other to form a structure containing a single-stranded region and a double-stranded region. Fluorescence signals are collected at the denaturation temperature (95 DEG C) and the annealing / extension temperature (60 DEG C) of each cycle of PCR amplification, respectively, two groups of independent amplification curves are generated in the same fluorescence channel, and two targets are distinguished, respectively, without the need of melting curve analysis. The application also provides related detection systems, kits and detection methods, and is suitable for simultaneous detection of various target nucleic acids.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, specifically to fluorescence quantitative PCR multiplex nucleic acid detection, and more specifically to a nucleic acid complex, a nucleic acid detection system, a nucleic acid detection kit, and a multiplex nucleic acid detection method based on temperature-dependent fluorescence signal acquisition. Background Technology

[0002] Quantitative real-time PCR (qPCR) technology has become the mainstream technique in nucleic acid detection due to its ease of operation, high sensitivity, ability to amplify and detect in a closed system in real time, and significantly reduced risk of contamination. This technology allows for multiplex amplification by designing primers and probes targeting different genes within the same reaction system. Currently, qPCR probes include TaqMan probes, molecular beacons, and Scorpions probes, all of which possess high analytical sensitivity. However, limited by the number of fluorescence channels in existing instruments, most qPCR instruments are equipped with only four fluorescent dye channels, thus allowing for the simultaneous detection of a maximum of four different targets in the same reaction tube. To overcome this limitation, some approaches have expanded the number of targets detectable per tube to ten by improving the dye system and utilizing the instrument's ability to interpret different wavelengths. However, the cost of the additional dyes and supporting instruments required far exceeds conventional levels, resulting in high promotion costs.

[0003] In multiplex detection schemes based on a single fluorescence channel, melting curve-based methods distinguish different targets by designing probes with different Tm values. However, when the number of targets increases, the difference in melting peak temperature between different probes may only be about 1°C, requiring extremely high reagent stability and instrument accuracy. Furthermore, the close proximity of melting peak temperatures can easily lead to false positives, presenting significant technical drawbacks. In addition, this method requires an extra melting curve analysis step after PCR amplification, increasing the operational process and detection time. Exposure of PCR products to an open environment also poses a risk of cross-contamination. While reverse dot blot hybridization and flow cytometry can achieve high-throughput multiplex detection, the former is difficult to automate and requires extremely high operator skill and laboratory conditions. The latter's detection signal reflects the concentration of PCR products rather than the original nucleic acid concentration, showing low correlation with the original viral load. Moreover, the hybridization of PCR products with probes requires denaturation, and the high-temperature step is not conducive to automation. Therefore, there is still a need for a simple, rapid, and accurate multiplex nucleic acid amplification and detection method that utilizes existing fluorescent dyes and conventional instrument performance, eliminates the need for melting curve post-processing, and is easy to operate. Summary of the Invention

[0004] The technical problem this invention aims to solve is that existing quantitative real-time PCR (qPCR) techniques are limited by the number of fluorescence channels, typically allowing only one target nucleic acid to be detected within the same channel. Furthermore, multiplex detection methods based on melting curves suffer from drawbacks such as adjacent melting peak temperature differences of only about 1°C, a high false-positive probability, the need for additional melting curve analysis after PCR amplification, increased operational procedures, and increased risk of contamination. Existing technologies lack a simple and rapid multiplex nucleic acid amplification and detection method that does not require increasing the number of fluorescence channels or modifying the instrument, eliminates the need for melting curve post-processing, and is easy to operate.

[0005] To address the aforementioned technical problems, the present invention provides the following technical solutions.

[0006] On one hand, the present invention provides a nucleic acid complex, which is composed of a first nucleic acid, a second nucleic acid, and a third nucleic acid; the first nucleic acid is simultaneously modified with a fluorescent group and a quencher group; the second nucleic acid is simultaneously modified with a fluorescent group and a quencher group, and its 3' end is phosphorylated; the third nucleic acid is modified with a quencher group only at its 5' end; the second nucleic acid and the third nucleic acid form a structure containing both single-stranded and double-stranded regions through complementary base pairing; wherein, the first nucleic acid is complementary to the target nucleic acid, the third nucleic acid is complementary to a portion of the second nucleic acid, and a portion of the second nucleic acid is complementary to the target nucleic acid sequence.

[0007] In one embodiment, the quenching group of the first nucleic acid is modified at the 5' end, the fluorescent group is modified internally, and the 3' end is left unmodified; both the fluorescent group and the quenching group of the second nucleic acid are modified internally. This design of modification positions allows the first and second nucleic acids to exhibit different fluorescence states under different temperature conditions, forming the structural basis for achieving temperature-dependent dual-signal processing.

[0008] In one embodiment, the fluorescent group is selected from one or more of FAM, VIC, ROX, and CY5; the quenching group is selected from one or more of BHQ, TAMRA, and DABCYL.

[0009] In one embodiment, the first nucleic acid consists of 30 to 45 nucleotides; the second nucleic acid consists of 30 to 45 nucleotides; and the third nucleic acid consists of 15 to 25 nucleotides.

[0010] In one embodiment, the molar ratio of the first nucleic acid, the second nucleic acid, and the third nucleic acid is 1-10:1-5:1-10, preferably 1-5:1-3:1-5, for example 2:1:2.

[0011] In one embodiment, the first nucleic acid has a nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:7; and the third nucleic acid has a nucleotide sequence as shown in SEQ ID NO:6 or SEQ ID NO:12.

[0012] On the other hand, the present invention provides a nucleic acid detection system, which includes the above-mentioned nucleic acid complex and DNA polymerase.

[0013] In one embodiment, the nucleic acid detection system further includes a PCR reaction solution, which contains 10×PCR buffer, 0.05–1 mM dNTP, and 1–5 mM MgCl2.

[0014] In one embodiment, the nucleic acid detection system further comprises an upstream primer and a downstream primer for amplifying the target nucleic acid. The primers may have nucleotide sequences as shown in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or nucleotide sequences as shown in SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

[0015] On the other hand, the present invention provides a nucleic acid detection kit, which includes the above-mentioned nucleic acid detection system. In one embodiment, the nucleic acid detection kit includes: a first container and the nucleic acid detection system located within the first container; it may also include a second container and a DNA polymerase located within the second container; it may also include a third container and a PCR reaction solution located within the third container; and / or it may also include a fourth container and primers located within the fourth container; wherein the first container, the second container, the third container, and / or the fourth container are the same or different containers.

[0016] On the other hand, the present invention provides a nucleic acid detection method, the method comprising the following steps:

[0017] (s1) Provide a premix, a sample to be tested, and DNA polymerase, wherein the premix contains a nucleic acid complex composed of a first nucleic acid, a second nucleic acid, and a third nucleic acid, the structure of which is as described above;

[0018] (s2) The premixed solution, the sample to be tested, and the DNA polymerase are mixed and then subjected to PCR amplification;

[0019] (s3) In each cycle of PCR amplification, fluorescence signals were collected once at the denaturation temperature step and once at the annealing / extension temperature step.

[0020] (s4) Plot the first amplification curve based on the fluorescence value collected at the denaturation temperature step and the cycle number, and plot the second amplification curve based on the fluorescence value collected at the annealing / extension temperature step and the cycle number. Two sets of amplification curves are generated in the same fluorescence channel in the same PCR reaction.

[0021] (s5) Based on the two sets of amplification curves, the two targets are interpreted respectively. The amplification curves generated at different temperatures correspond to the targets detected by probes with different modification methods.

[0022] In one embodiment, the temperature of the denaturation temperature step in step (s3) is 95°C, and the temperature of the annealing / stretching temperature step is 60°C.

[0023] In one embodiment, the PCR amplification program is as follows: for DNA template, pre-denaturation at 95°C for 2 minutes per cycle, followed by 40-50 cycles of 95°C for 15 seconds, 60°C for 20 seconds, and 72°C for 15 seconds; for RNA template, reverse transcription at 50°C for 10 minutes per cycle, pre-denaturation at 95°C for 2 minutes per cycle, followed by 40-50 cycles of 95°C for 15 seconds, 60°C for 20 seconds, and 72°C for 15 seconds.

[0024] In one embodiment, the premix further comprises upstream and downstream primers for amplifying the target nucleic acid.

[0025] In one embodiment, the sample to be tested is subjected to reverse transcription; step (s3) includes heating at 95°C for 15 seconds and collecting fluorescence signals, cooling to 60°C and collecting fluorescence signals.

[0026] The present invention also provides another nucleic acid detection method, in which the above-mentioned nucleic acid detection system or the above-mentioned nucleic acid detection kit is used to perform nucleic acid amplification reaction on the sample to be tested, and fluorescence signals are collected and analyzed at 95℃ and 60℃ respectively to obtain the detection results.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] First, by designing a ternary nucleic acid complex and utilizing the existing denaturation temperature step (95℃) and annealing / extension temperature step (60℃) in the PCR amplification cycle to collect fluorescence signals, two independent amplification curves can be generated simultaneously in the same reaction tube and the same fluorescence channel, thereby achieving simultaneous detection of two target nucleic acids without increasing the number of fluorescence channels or modifying the existing quantitative PCR instrument.

[0029] Secondly, the detection method of the present invention collects and interprets fluorescence signals in real time during PCR amplification, eliminating the need for additional melting curve analysis after PCR amplification, thus shortening the detection time and reducing the risk of PCR products being exposed to an open environment.

[0030] Third, since the amplification signals of the two targets are generated independently at different temperatures, and the signals at their respective temperatures do not interfere with each other, the probability of false positives due to excessively close melting peak temperatures is reduced compared to multiple detection methods based on melting curves. Sensitivity comparison data from Example 6 (Table 12) shows that in the detection of HPV16 and HPV31, the method of this invention can detect all samples at 100 copies, while the comparative method shows false negatives at 100 copies; at 50 copies, the method of this invention can detect HPV16 samples 16-1 and 16-2 and HPV31 sample 31-1, while the comparative method shows multiple false negatives.

[0031] Fourth, the method of the present invention uses a common DNA polymerase, the operation steps are simple, and it can be used on most conventional real-time PCR instruments, thus having good applicability. Attached Figure Description

[0032] Figure 1 A schematic diagram illustrating the reaction mechanism of the ternary nucleic acid complex at 60°C and 95°C, as provided in an embodiment of the present invention.

[0033] Figure 2 The real-time fluorescence PCR amplification curves for simultaneous detection of HPV16 and HPV31 using the same fluorescence channel provided in this embodiment of the invention are shown in the figure above, where the HPV16 amplification curve was collected at 60℃ and the HPV31 amplification curve was collected at 95℃.

[0034] Figure 3 The real-time fluorescence PCR amplification curve of influenza A virus (IFA) at a collection temperature of 60°C is provided for an embodiment of the present invention.

[0035] Figure 4 This is a real-time fluorescent PCR amplification curve of influenza B virus (IFB) at a collection temperature of 95°C, provided as an embodiment of the present invention. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Methods in the following embodiments that do not specify specific conditions are generally performed under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0037] Terminology Definition

[0038] In this article, "first nucleic acid" refers to a nucleic acid strand that is simultaneously modified with a fluorescent group and a quencher group, and is complementary to the target nucleic acid. The quencher group is modified at the 5' end, the fluorescent group is modified internally, and the 3' end is left unmodified. During PCR amplification, the first nucleic acid binds complementary to the target nucleic acid and is extended by DNA polymerase. During extension, the distance between the fluorescent group and the quencher group increases, and a fluorescent signal is released.

[0039] "Second nucleic acid" refers to a nucleic acid chain that is simultaneously modified with a fluorescent group and a quencher group, and whose 3' end is phosphorylated. Both the fluorescent group and the quencher group are internally modified. The 3' phosphorylation prevents the second nucleic acid from being directly extended by DNA polymerase. A portion of the second nucleic acid sequence is complementary to the target nucleic acid sequence. Under the action of the 5'→3' exonuclease activity of DNA polymerase, the complementary portion of the second nucleic acid is cleaved, causing the internal quencher group of the second nucleic acid to detach along with the cleaved fragment, thereby altering the fluorescence state of the second nucleic acid.

[0040] "Third nucleic acid" refers to a nucleic acid chain with a quenching group modified only at its 5' end. It forms a double-stranded region with a portion of the second nucleic acid through base complementarity, while retaining a single-stranded region, thus creating a nucleic acid complex structure containing both single-stranded and double-stranded regions. At an annealing / stretching temperature of 60°C, the quenching group at the 5' end of the third nucleic acid approaches the fluorescent group of the second nucleic acid through the double-stranded region, thereby quenching the fluorescence signal of the second nucleic acid. At a denaturing temperature of 95°C, the double strands between the second and third nucleic acids unwind, the quenching group of the third nucleic acid moves away from the fluorescent group of the second nucleic acid, and the fluorescence signal of the second nucleic acid is released.

[0041] Reaction mechanism of ternary nucleic acid complex

[0042] The temperature-dependent fluorescence signal mechanism of the ternary nucleic acid complex in the PCR amplification process of this invention is as follows: Figure 1 As shown.

[0043] In a detection system containing target nucleic acid (DNA template), each cycle of PCR amplification includes a denaturation temperature step (95℃) and an annealing / extension temperature step (60℃). Fluorescence signals are collected at the two temperature steps, and two independent amplification curves can be generated in the same fluorescence channel, corresponding to the detection results of two different target nucleic acids.

[0044] In the annealing / extension step at 60℃: the first nucleic acid binds to its complementary target nucleic acid (first target nucleic acid) and is extended by DNA polymerase. During extension, the distance between the fluorophore and the 5' quencher of the first nucleic acid increases, generating a fluorescent signal. Simultaneously, the complementary portion of the second nucleic acid to the second target nucleic acid is cleaved by the 5'→3' exonuclease activity of DNA polymerase. The quencher within the second nucleic acid is detached along with the cleaved fragment. However, the third nucleic acid and the second nucleic acid still maintain a double-stranded region. The quencher at the 5' end of the third nucleic acid continues to quench the fluorescent signal of the second nucleic acid, and the second nucleic acid does not fluoresce at 60℃. Therefore, the fluorescent signal collected at 60℃ originates only from the first nucleic acid, reflecting the amplification of the first target nucleic acid.

[0045] At the 95°C denaturation temperature step: all complementary double strands dissolve, the first nucleic acid extension product becomes single-stranded, the fluorophore of the first nucleic acid and the 5' quencher group move closer together again, and the fluorescence signal decreases; the double-stranded region between the second and third nucleic acids unwinds, the 5' quencher group of the third nucleic acid moves away from the fluorophore of the second nucleic acid, and the fluorescence signal of the second nucleic acid is released. Therefore, the fluorescence signal collected at 95°C originates from the second nucleic acid and reflects the amplification of the second target nucleic acid.

[0046] In summary, by collecting fluorescence values ​​at 60℃ and 95℃ in the same PCR reaction tube and the same fluorescence channel, two independent amplification curves can be plotted: the amplification curve collected at 60℃ corresponds to the target detected by the first nucleic acid, and the amplification curve collected at 95℃ corresponds to the target detected by the second nucleic acid. This achieves simultaneous detection of dual targets in a single channel without the need for additional melting curve analysis after PCR amplification.

[0047] General procedures for nucleic acid testing systems

[0048] The nucleic acid detection system of the present invention comprises a ternary nucleic acid complex and a DNA polymerase, and may also include a PCR reaction solution and upstream and downstream primers for amplifying the target nucleic acid.

[0049] The PCR reaction solution may contain 10×PCR buffer, 0.05–1 mM dNTPs, and 1–5 mM MgCl2. In one embodiment, each dNTP is 0.2 mM and the MgCl2 is 1.5 mM.

[0050] The concentration of the first nucleic acid in the detection system can be 1 nM to 500 mM, preferably 10 nM to 50 mM, more preferably 50 to 500 nM, for example, about 200 nM. The concentration of the second nucleic acid can be 1 nM to 300 mM, preferably 10 nM to 50 mM, more preferably 20 to 500 nM, for example, about 50 nM. The concentration of the third nucleic acid can be 1 nM to 500 mM, preferably 10 nM to 50 mM, more preferably 50 to 500 nM, for example, about 100 nM. The molar ratio of the first, second, and third nucleic acids can be 1 to 10: 1 to 5: 1 to 10, preferably 1 to 5: 1 to 3: 1 to 5, for example, 2: 1: 2.

[0051] The amount of DNA polymerase used can be 0.5–5 U, preferably 0.5–3 U, more preferably 0.5–2 U, for example, about 1 U. The concentrations of the upstream and downstream primers in the detection system can each be independently 50–500 nM, preferably 100–400 nM, more preferably 150–300 nM, for example, about 200 nM.

[0052] For DNA templates, the PCR amplification program can be set as follows: one cycle of 95℃ pre-denaturation for 10 minutes; 40-50 cycles of 95℃ for 15 seconds, 60℃ for 20 seconds, and 72℃ for 15 seconds; fluorescence signals are collected at the 95℃ and 60℃ steps of each cycle. For RNA templates, one cycle of 50℃ reverse transcription for 10 minutes and one cycle of 95℃ pre-denaturation for 2 minutes can be added before the above program.

[0053] Example 1: Simultaneous detection of HPV16 and HPV31 using a ternary nucleic acid complex

[0054] This embodiment demonstrates the feasibility of the method by designing a ternary nucleic acid complex targeting HPV16 and HPV31, and simultaneously detecting HPV DNA of the two types using a single fluorescence channel in the same reaction tube.

[0055] 1. Design of the first nucleic acid

[0056] A first nucleic acid (SEQ ID NO:1, HPV16-F) and its corresponding primer (SEQ ID NO:2, HPV16-R) were designed targeting highly specific sequences in the HPV16 genome. The 5' end of the first nucleic acid (SEQ ID NO:1) was modified with a quencher group BHQ1, the interior was modified with a fluorescent group FAM, and the 3' end remained unmodified. The primer sequences are shown in Table 1.

[0057] Table 1. First nucleic acid and primer sequences for HPV detection

[0058]

[0059] 2. Design of the second and third nucleic acids

[0060] We designed a second nucleic acid (SEQ ID NO:5, HPV31-Probe) and a third nucleic acid (SEQ ID NO:6, HPV31-Blocker) targeting highly specific sequences in the HPV31 genome, as well as HPV31 amplification primers (SEQ ID NO:3, HPV31-F; SEQ ID NO:4, HPV31-R).

[0061] The HPV31 primer sequences are shown in Table 2a, the second nucleic acid sequence is shown in Table 2b, and the third nucleic acid sequence is shown in Table 2c.

[0062] Table 2a HPV31 primer sequences

[0063]

[0064] Table 2b Second Nucleic Acid Sequence

[0065]

[0066] Table 2c Third Nucleic Acid Sequence

[0067]

[0068] The second nucleic acid, SEQ ID NO:5, is internally modified with a FAM fluorescent group and a ZEN quencher group, and its 3' end is phosphorylated. The 5' end of the third nucleic acid, SEQ ID NO:6, is modified with a BHQ1 quencher group. The second and third nucleic acids form a complex structure containing single-stranded and double-stranded regions through complementary base pairing. The molar ratio of the three in the detection system is first nucleic acid: second nucleic acid: third nucleic acid = 2:1:2.

[0069] 3. PCR detection system

[0070] A nonionic detergent combined with a high-temperature environment was used to lyse the HPV outer membrane proteins, releasing the DNA. Simultaneously, Chelex-100 was used to remove impurities such as metal ions that inhibit PCR, thereby improving detection efficiency. The optimized PCR system is shown in Table 3a, and the PCR reaction procedure is shown in Table 3b.

[0071] Table 3a PCR system

[0072]

[0073] Table 3b PCR reaction procedure

[0074]

[0075] 4. Amplification curve analysis

[0076] On a real-time PCR instrument, fluorescence signals from the FAM channel were collected at 95°C and 60°C in each PCR cycle. Amplification curves were plotted for the fluorescence values ​​collected at 95°C and the cycle number (corresponding to HPV31 detection), and for the fluorescence values ​​collected at 60°C and the cycle number (corresponding to HPV16 detection). Two independent amplification curves were generated in the same reaction tube and the same fluorescence channel.

[0077] 5. Results

[0078] like Figure 2 As shown, by designing primers, first nucleic acid, second nucleic acid, and third nucleic acid for two HPV types, HPV16 and HPV31, the fluorescence signals of the two types are collected at 60℃ and 95℃ respectively, emitting light only at their respective temperatures. This reduces mutual interference between different types and lowers the probability of false positives. The method of this invention can simultaneously detect two HPV types using a single fluorescence channel.

[0079] Example 2: Simultaneous detection of influenza A and influenza B viruses using a ternary nucleic acid complex

[0080] This embodiment demonstrates the feasibility of the method of the present invention in RNA template detection by designing a ternary nucleic acid complex targeting influenza A virus (INFA) and influenza B virus (INFB) and simultaneously detecting the RNA of the two influenza viruses using a single fluorescent channel in the same reaction tube.

[0081] 1. Primer and probe design

[0082] The genome sequences of influenza A and influenza B viruses were downloaded from the NCBI database for comparison. Primers were designed based on highly specific sequences in the genomes, and PCR was performed in the same tube using a single fluorescent channel to simultaneously detect influenza A and influenza B viruses. The primers, first nucleic acid, second nucleic acid, and third nucleic acid sequences used in this example are shown in Table 4.

[0083] Table 4 Primer and probe sequences

[0084]

[0085] The first nucleic acid, SEQ ID NO:7 (INFA), has a quencher group BHQ1 modified at its 5' end and a fluorescent group VIC modified internally. Its 3' end remains unmodified, and it is complementary to the target sequence of influenza A virus. The second nucleic acid, SEQ ID NO:11 (INFB), has a fluorescent group VIC and a quencher group modified internally, and its 3' end is phosphorylated. A portion of its sequence is complementary to the target sequence of influenza B virus. The third nucleic acid, SEQ ID NO:12 (INFB), has a quencher group BHQ1 modified at its 5' end, and it forms a complex structure containing single-stranded and double-stranded regions with a portion of the second nucleic acid through base complementarity pairing. The molar ratio of the three in the detection system is: first nucleic acid: second nucleic acid: third nucleic acid = 2:1:2.

[0086] 2. PCR reaction system

[0087] RNA / DNA of influenza A and influenza B viruses were extracted using the magnetic bead method. The optimized PCR system is shown in Table 5a, and the PCR reaction procedure is shown in Table 5b.

[0088] Table 5a PCR system

[0089]

[0090] Table 5b PCR reaction procedure

[0091]

[0092] 3. Amplification curve analysis

[0093] On a real-time PCR instrument, fluorescence signals from the VIC channel were collected at 95°C and 60°C in each PCR cycle. Based on the design of primers, the first nucleic acid, the second nucleic acid, and the third nucleic acid, fluorescence signals were collected at 95°C and 60°C for detection.

[0094] 4. Results

[0095] like Figure 3 As shown, fluorescent amplification signals of influenza A virus (INFA) can be collected at a collection temperature of 60℃; as Figure 4 As shown, fluorescent amplification signals of influenza B virus (INFB) can be collected at a collection temperature of 95°C. The fluorescent signals of the two types are collected at 60°C and 95°C respectively, emitting light only at their respective temperatures, reducing mutual interference between different types and lowering the probability of false positives. This invention's method can simultaneously detect influenza A and influenza B viruses using a single fluorescence channel.

[0096] Example 3 (Comparative Example): Detection of HPV16 and HPV31 using conventional TaqMan probes

[0097] This example is a comparative example. Conventional TaqMan probes (without ternary nucleic acid complex structure) were used to detect HPV16 and HPV31 for comparison with Example 1.

[0098] 1. Primer and probe design

[0099] The same HPV16 first nucleic acid (SEQ ID NO:1) and HPV16 primers (SEQ ID NO:2) and HPV31 primers (SEQ ID NO:3, SEQ ID NO:4) as in Example 1 were used. However, a conventional TaqMan probe (SEQ ID NO:13, HPV31-Probe) containing only 5' FAM and 3' ZEN modifications was used instead of the second / third nucleic acid ternary structure in Example 1. SEQ ID NO:13 differs from SEQ ID NO:5 in Example 1 in that it does not contain 3' phosphorylation modification and does not form a complex with the third nucleic acid. The sequences are shown in Table 6.

[0100] Table 6 Comparative Primer and Probe Sequences

[0101]

[0102] 2. PCR detection system

[0103] Nonionic detergents and high-temperature environment were used to lyse HPV outer membrane proteins, and Chelex-100 was used to remove impurities that inhibited PCR to improve detection efficiency. The PCR system is shown in Table 7a, and the PCR reaction procedure is shown in Table 7b.

[0104] Table 7a PCR system

[0105]

[0106] Table 7b PCR reaction procedure

[0107]

[0108] 3. Amplification curve analysis

[0109] On a real-time PCR instrument, fluorescence signals from the FAM channel were collected and detected at the 95°C and 60°C steps of each PCR cycle.

[0110] 4. Results

[0111] Using the conventional TaqMan probe method, fluorescence signals for both HPV16 and HPV31 types can be collected at 95℃ and 60℃, respectively—fluorescence signals for HPV31 amplification can be collected at 95℃, while fluorescence signals for HPV16 and HPV31 amplification can be collected at 60℃. Because conventional probes generate signals at both temperatures, there is mutual interference between different types, and conventional probe design methods cannot achieve the goal of simultaneously and specifically detecting two HPV types using a single fluorescence channel.

[0112] Example 4 (Comparative Example): Detection of Influenza A and Influenza B Viruses using Conventional Probes

[0113] This example is a comparative example, using conventional probes (without ternary nucleic acid complex structure) to detect influenza A and influenza B viruses, in order to compare with Example 2.

[0114] 1. Primer and probe design

[0115] The same influenza A virus first nucleic acid (SEQ ID NO:7) and primers (SEQ ID NO:8) and influenza B virus primers (SEQ ID NO:9, SEQ ID NO:10) as in Example 2 were used. The second / third nucleic acid ternary structure in Example 2 was replaced with a conventional probe (SEQ ID NO:14, probe INFB) containing only 5'VIC modification and 3'ZEN modification. The sequences are shown in Table 8.

[0116] Table 8 Comparative Primer and Probe Sequences

[0117]

[0118] 2. PCR reaction system

[0119] RNA was extracted from influenza A and influenza B viruses using the magnetic bead method. The PCR system is shown in Table 9a, and the PCR reaction procedure is shown in Table 9b.

[0120] Table 9a PCR System

[0121]

[0122] Table 9b PCR reaction procedure

[0123]

[0124] 3. Amplification curve analysis

[0125] On a real-time PCR instrument, fluorescence signals from the VIC channel were collected and detected at the 95°C and 60°C steps of each PCR cycle.

[0126] 4. Results

[0127] Using conventional probe methods, both influenza A and influenza B viruses can be detected with fluorescence signals at 95℃ and 60℃, respectively—95℃ yields amplified fluorescence signals for influenza B virus, while 60℃ yields fluorescence signals for both influenza A and influenza B viruses. Therefore, conventional probe design methods cannot use a single fluorescence channel to simultaneously and specifically detect both influenza A and influenza B viruses.

[0128] Example 5 (Comparative Example): A Multiplex HPV Detection Method Based on Melting Curves

[0129] This embodiment serves as a comparative example, employing conventional melting curve analysis methods to perform multiple detections of HPV16 and HPV31, thereby illustrating the limitations of the melting curve method.

[0130] 1. Primer and probe sequences

[0131] Conventional multiplex detection methods based on melting curves design degenerate primers on sequences that have both homology and specificity for each HPV type gene, and design probes on type-specific sequences. The primer and probe sequences used in this embodiment are shown in Table 10.

[0132] Table 10 Comparative Primer and Probe Sequences

[0133]

[0134] 2. PCR detection system

[0135] HPV outer membrane proteins were lysed using a nonionic detergent and a high-temperature environment, and impurities that inhibited PCR were removed using Chelex-100. The optimized primer and probe concentrations and PCR system are shown in Table 10a, and the PCR reaction program is shown in Table 10b.

[0136] Table 10a PCR system

[0137]

[0138] Table 10b PCR reaction procedure

[0139]

[0140] 3. Melting curve analysis

[0141] After PCR amplification, melting curve analysis was performed. The reaction procedure is shown in Table 11.

[0142] Table 11 Reaction Procedure for Melting Curve Analysis

[0143]

[0144] 4. Results

[0145] The melting curve height in each result is compared with that in the negative control; a change in melting curve height exceeding 10% is considered positive. Because the melting peak temperatures of HPV16 and HPV31 probes are very similar (approximately 1°C), this method requires extremely high reagent stability and instrument precision. Furthermore, the close proximity of the melting peak temperatures can easily lead to false positives, necessitating an additional melting curve analysis step after PCR amplification, thus increasing the operational process and time.

[0146] Example 6: Sensitivity Comparison of the Invention Method and Conventional Methods

[0147] This embodiment uses standards containing HPV16 and HPV31 plasmids and performs detection using the methods described in Example 1 (method of the present invention) and Example 3 (conventional method) to compare the detection sensitivity of the two methods.

[0148] Sample setup: Standards containing 10,000, 1,000, 100, 50, and 25 copies of HPV16 (samples 16-1 and 16-2) and HPV31 (samples 31-1 and 31-2) plasmids, and water as a negative control. The results are shown in Table 12.

[0149] Table 12. Sensitivity Comparison between the Method of the Present Invention (Example 1) and the Conventional Method (Example 3)

[0150]

[0151] As shown in Table 12, when the target count was 100 copies, the conventional method (Example 3) could not detect HPV31 sample 31-2, resulting in false negatives, while the method of the present invention (Example 1) could detect all samples. At 50 copies, the method of the present invention could detect HPV16 samples 16-1 and 16-2, and HPV31 sample 31-1, while the conventional method produced false negatives for HPV16 samples 16-2 and HPV31 samples 31-2; HPV31 sample 31-2 was not detected by either method at 50 copies. At 25 copies, neither method detected any samples. In the target-free negative control, both methods maintained high specificity and no false positives occurred.

[0152] The above results demonstrate that, compared to conventional probe methods (Example 3), the method of the present invention (Example 1) has higher detection sensitivity, detecting all test samples at 100 copies and most samples at 50 copies, with a detection limit of approximately 50 copies (based on the aforementioned test samples). Compared to Example 3, the method of the present invention also achieves closed-tube detection, eliminating the need for additional steps such as melting curve analysis after PCR, thus shortening the detection time and reducing the risk of contamination from amplification products.

[0153] Example 7: A ternary nucleic acid complex with FAM as the fluorescent group and BHQ as the quencher group

[0154] This embodiment provides a ternary nucleic acid complex, which is composed of a first nucleic acid, a second nucleic acid, and a third nucleic acid. The first nucleic acid is modified with both a fluorescent group and a quencher group; the quencher group is modified at the 5' end, the fluorescent group is modified internally, and the 3' end is unmodified. The second nucleic acid is also modified with both a fluorescent group and a quencher group; both are modified internally, and the 3' end is phosphorylated. The third nucleic acid is modified only with a quencher group at the 5' end. The second and third nucleic acids form a structure containing both single-stranded and double-stranded regions through complementary base pairing. Specifically, the first nucleic acid is complementary to the first target nucleic acid, the third nucleic acid is complementary to a portion of the second nucleic acid, and a portion of the second nucleic acid is complementary to the second target nucleic acid sequence. In this embodiment, the fluorescent group is FAM, and the quencher group is BHQ.

[0155] Example 8: A ternary nucleic acid complex with ROX and CY5 fluorescent groups and TAMRA and DABCYL quenching groups.

[0156] This embodiment provides a ternary nucleic acid complex, which is composed of a first nucleic acid, a second nucleic acid, and a third nucleic acid. The first nucleic acid is modified with both a fluorescent group and a quencher group; the quencher group is modified at the 5' end, the fluorescent group is modified internally, and the 3' end is unmodified. The second nucleic acid is also modified with both a fluorescent group and a quencher group; both are modified internally, and the 3' end is phosphorylated. The third nucleic acid is modified only with a quencher group at the 5' end. The second and third nucleic acids form a structure containing both single-stranded and double-stranded regions through complementary base pairing. Specifically, the first nucleic acid is complementary to the first target nucleic acid, the third nucleic acid is complementary to a portion of the second nucleic acid, and a portion of the second nucleic acid is complementary to the second target nucleic acid sequence. In this embodiment, the fluorescent groups are ROX and CY5, and the quencher groups are TAMRA and DABCYL.

[0157] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing description of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A nucleic acid complex, characterized in that, The nucleic acid complex is composed of a first nucleic acid, a second nucleic acid, and a third nucleic acid; the first nucleic acid is modified with both a fluorescent group and a quencher group; the second nucleic acid is modified with both a fluorescent group and a quencher group, and its 3' end is phosphorylated; the third nucleic acid is modified with a quencher group only at its 5' end; the second nucleic acid and the third nucleic acid form a structure containing both single-stranded and double-stranded regions through complementary base pairing; wherein, the first nucleic acid is complementary to the target nucleic acid, the third nucleic acid is complementary to a portion of the second nucleic acid, and a portion of the second nucleic acid is complementary to the target nucleic acid sequence.

2. The nucleic acid complex according to claim 1, characterized in that, The first nucleic acid has a quenching group modified at the 5' end, a fluorescent group modified internally, and no modification at the 3' end; the second nucleic acid has both a fluorescent group and a quenching group modified internally.

3. The nucleic acid complex according to claim 1, characterized in that, The fluorescent group is selected from one or more of FAM, VIC, ROX, and CY5; the quenching group is selected from one or more of BHQ, TAMRA, and DABCYL.

4. The nucleic acid complex according to claim 1, characterized in that, The first nucleic acid consists of 30 to 45 nucleotides; the second nucleic acid consists of 30 to 45 nucleotides; and the third nucleic acid consists of 15 to 25 nucleotides.

5. The nucleic acid complex according to claim 1, characterized in that, The molar ratio of the first nucleic acid, the second nucleic acid, and the third nucleic acid is 1–10: 1–5: 1–10.

6. The nucleic acid complex according to claim 1, characterized in that, The first nucleic acid has a nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:7; the third nucleic acid has a nucleotide sequence as shown in SEQ ID NO:6 or SEQ ID NO:

12.

7. A nucleic acid detection system, characterized in that, The nucleic acid detection system comprises the nucleic acid complex and DNA polymerase as described in any one of claims 1 to 6.

8. The nucleic acid detection system according to claim 7, characterized in that, The nucleic acid detection system also includes a PCR reaction solution, which contains 10×PCR buffer, 0.05–1 mM dNTP, and 1–5 mM MgCl2.

9. The nucleic acid detection system according to claim 7, characterized in that, The nucleic acid detection system also includes upstream and downstream primers for amplifying the target nucleic acid.

10. The nucleic acid detection system according to claim 9, characterized in that, The primer has a nucleotide sequence as shown in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, or the primer has a nucleotide sequence as shown in SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:

10.

11. A nucleic acid detection kit, characterized in that, The nucleic acid detection kit comprises the nucleic acid detection system according to any one of claims 7 to 10.

12. The nucleic acid detection kit according to claim 11, characterized in that, The nucleic acid detection kit comprises: a first container and the nucleic acid detection system located within the first container; a second container and a DNA polymerase located within the second container; a third container and a PCR reaction solution located within the third container; and / or a fourth container and primers located within the fourth container; wherein the first container, the second container, the third container, and / or the fourth container are the same or different containers.

13. A nucleic acid detection method, characterized in that, The method includes the following steps: (s1) Provide a premix, a sample to be tested, and DNA polymerase, wherein the premix contains a nucleic acid complex composed of a first nucleic acid, a second nucleic acid, and a third nucleic acid, and the nucleic acid complex is the nucleic acid complex according to any one of claims 1 to 6; (s2) The premixed solution, the sample to be tested, and the DNA polymerase are mixed and then subjected to PCR amplification; (s3) In each cycle of PCR amplification, fluorescence signals were collected once at the denaturation temperature step and once at the annealing / extension temperature step. (s4) Plot the first amplification curve based on the fluorescence value collected at the denaturation temperature step and the cycle number, and plot the second amplification curve based on the fluorescence value collected at the annealing / extension temperature step and the cycle number. Two sets of amplification curves are generated in the same fluorescence channel in the same PCR reaction. (s5) Based on the two sets of amplification curves, the two targets are interpreted respectively. The amplification curves generated at different temperatures correspond to the targets detected by probes with different modification methods.

14. The nucleic acid detection method according to claim 13, characterized in that, The temperature of the denaturation temperature step in step (s3) is 95°C, and the temperature of the annealing / stretching temperature step is 60°C.

15. The nucleic acid detection method according to claim 13, characterized in that, The PCR amplification program is as follows: For DNA template, pre-denaturation at 95℃ for 2 minutes is performed as one cycle, followed by 40-50 cycles of 95℃ for 15 seconds, 60℃ for 20 seconds, and 72℃ for 15 seconds; For RNA template, reverse transcription at 50℃ for 10 minutes is performed as one cycle, pre-denaturation at 95℃ for 2 minutes is performed as one cycle, followed by 40-50 cycles of 95℃ for 15 seconds, 60℃ for 20 seconds, and 72℃ for 15 seconds.

16. The nucleic acid detection method according to claim 13, characterized in that, The premix also contains upstream and downstream primers for amplifying the target nucleic acid.

17. The nucleic acid detection method according to claim 13, characterized in that, The sample to be tested is reverse transcribed; step (s3) includes heating at 95°C for 15 seconds and collecting fluorescence signals, cooling to 60°C and collecting fluorescence signals.

18. A nucleic acid detection method, characterized in that, After performing nucleic acid amplification reaction on the sample to be tested using the nucleic acid detection system of any one of claims 7 to 10 or the nucleic acid detection kit of any one of claims 11 to 12, fluorescence signals are collected and analyzed at 95℃ and 60℃ respectively to obtain the detection results.

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