Multi-PCR (Polymerase Chain Reaction) detection nucleic acid composition for treponema pallidum and herpes simplex virus, detection product and application of detection product

Abstract

The invention discloses a multiplex PCR detection nucleic acid composition for treponema pallidum and herpes simplex virus, a detection product and application of the multiplex PCR detection nucleic acid composition and the detection product, and relates to the technical field of pathogen detection. According to the nucleic acid composition provided by the invention, four pathogen targets and one internal reference can be detected in the same reaction system by virtue of a fluorescent PCR instrument with four different fluorescent channels, and the nucleic acid composition has the technical advantages of high detection specificity, good typing effect and highly consistent detection results.

Description

Technical Field

[0001] This invention relates to the field of pathogen detection technology, and more specifically, to a nucleic acid composition for multiplex PCR detection of Treponema pallidum and herpes simplex virus, a detection product, and its application. Background Technology

[0002] Sexually transmitted diseases (STDs) are a class of specific infectious diseases, primarily transmitted through sexual contact. Numerous pathogens cause STDs, resulting in a wide variety of symptoms, varying in transmissibility, morbidity, and severity. This presents significant challenges to the diagnosis and treatment of STDs. Among the many symptoms caused by STDs, one category manifests as localized skin tissue defects, liquefaction, or necrosis, commonly referred to as ulcers. These symptoms differ depending on the pathogen, the course of the disease, and the specific details. The main pathogens causing these symptoms are Treponema pallidum (syphilis bacillus) and herpes simplex virus (HSV). HSV, in particular, is further divided into two subtypes based on the common sites of infection: HSV type I and HSV type II.

[0003] In recent years, a new virus, monkeypox, has been widely circulating in some areas. The typical characteristic of monkeypox is a rash, which usually appears 1-3 days after fever. The rash first appears on the face and then spreads to other parts of the body. The rash progresses from macules to papules, vesicles, pustules, and finally crusts. From the perspective of the external symptoms, it is similar to those caused by Treponema pallidum and herpes simplex virus; therefore, it is necessary to differentiate and distinguish between them.

[0004] Fluorescent PCR is a widely used molecular biology technique in nucleic acid detection. It utilizes fluorescently labeled probes or dyes to monitor DNA synthesis in real time during PCR amplification, thereby enabling qualitative or quantitative analysis of specific nucleic acid sequences. Fluorescent PCR can be used to detect and quantify various pathogens, such as viruses, bacteria, and parasites. The advantages of fluorescent PCR technology lie in its high sensitivity, high specificity, speed, and quantifiability, making it an indispensable tool in the field of molecular diagnostics. With technological advancements, the application scope of fluorescent PCR continues to expand. Furthermore, primers or probes with different fluorescent dyes can be designed for each target. These dyes have different excitation and emission wavelengths, allowing them to be distinguished in the same reaction, thus enabling the simultaneous detection of multiple pathogens.

[0005] However, the most widely used fluorescence PCR instruments currently available typically only contain four different fluorescence channels. For nucleic acid detection kits, when detecting pathogen targets, a host or exogenous detection sequence is usually added as an internal control to monitor the effectiveness of the reaction system. Therefore, using a fluorescence PCR instrument with four different fluorescence channels, if different fluorescent dyes or fluorescently labeled probes are used to distinguish targets, can only detect a maximum of three pathogen targets. A fluorescence PCR instrument with four different fluorescence channels cannot achieve the detection of four pathogen targets plus one internal control in the same reaction system.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a nucleic acid composition, detection product and application for multiplex PCR detection of Treponema pallidum and herpes simplex virus, so as to achieve the detection of four pathogen targets plus one internal control in the same reaction system using a fluorescence PCR instrument with four different fluorescence channels.

[0008] This invention is implemented as follows: In a first aspect, the present invention provides a nucleic acid composition for multiplex PCR detection of Treponema pallidum and herpes simplex virus, comprising: The first nucleic acid combination for detecting Treponema pallidum, as shown in SEQ ID NO.1-3, the third nucleic acid combination for detecting herpes simplex virus type I, as shown in SEQ ID NO.7-9, and the fourth nucleic acid combination for detecting herpes simplex virus type II, as shown in SEQ ID NO.10-12, wherein the nucleotides in the sequence shown in SEQ ID NO.12 are not locked nucleic acid modified or at least one nucleotide is locked nucleic acid modified; The sequences shown in SEQ ID NO.3, SEQ ID NO.9, and SEQ ID NO.12 have a fluorescent reporter group at the 5' end and a fluorescent quencher group at the 3' end; The sequence shown in SEQ ID NO. 9 has the same or similar spectral characteristics at its 5' end as the sequence shown in SEQ ID NO. 12, and the sequence shown in SEQ ID NO. 9 has the same or similar spectral characteristics at its 5' end as the sequence shown in SEQ ID NO. 12.

[0009] Secondly, the present invention provides a nucleic acid composition for multiplex PCR detection of monkeypox virus, Treponema pallidum, and herpes simplex virus, comprising: The first nucleic acid combination for detecting Treponema pallidum, as shown in SEQ ID NO. 1-3; the second nucleic acid combination for detecting monkeypox virus, as shown in SEQ ID NO. 4-6; the third nucleic acid combination for detecting herpes simplex virus type I, as shown in SEQ ID NO. 7-9; and the fourth nucleic acid combination for detecting herpes simplex virus type II, as shown in SEQ ID NO. 10-12, wherein the nucleotides in the sequence shown in SEQ ID NO. 12 are not locked nucleic acid modified or at least one nucleotide is locked nucleic acid modified; The sequences shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, and SEQ ID NO.12 have a fluorescent reporter group at the 5' end and a fluorescent quencher group at the 3' end; The sequence shown in SEQ ID NO. 9 has the same or similar spectral characteristics at its 5' end as the sequence shown in SEQ ID NO. 12, and the sequence shown in SEQ ID NO. 9 has the same or similar spectral characteristics at its 5' end as the sequence shown in SEQ ID NO. 12.

[0010] Thirdly, the present invention provides a nucleic acid composition for multiplex PCR detection of Treponema pallidum and herpes simplex virus or a multiplex PCR detection product for monkeypox virus, Treponema pallidum and herpes simplex virus, comprising the above-mentioned nucleic acid composition for multiplex PCR detection of monkeypox virus, Treponema pallidum and herpes simplex virus.

[0011] Fourthly, the present invention provides a PCR premix comprising the above-mentioned multiplex PCR detection nucleic acid composition for Treponema pallidum and herpes simplex virus or a multiplex PCR detection nucleic acid composition for monkeypox virus, Treponema pallidum and herpes simplex virus; wherein the final concentration ratio of the primer sequences shown in SEQ ID NO. 1-2 to the probe sequences shown in SEQ ID NO. 3 in the PCR premix is ​​1:1-10:1. In the PCR premix, the final concentration ratio of the primer sequences shown in SEQ ID NO. 1-2 to the probe sequence shown in SEQ ID NO. 3 is 1:1-10:1; the final concentration ratio of the primer sequences shown in SEQ ID NO. 4-5 to the probe sequence shown in SEQ ID NO. 6 is 1:1-10:1; the final concentration ratio of the primer sequences shown in SEQ ID NO. 7 and SEQ ID NO. 8 is 2:1-10:1; and the final concentration ratio of the primer sequence shown in SEQ ID NO. 8 to the probe sequence shown in SEQ ID NO. 9 is 1:10-1:2. The final concentration ratio of the primer sequences shown in SEQ ID NO.10 and SEQ ID NO.11 is 2:1-10:1; the final concentration ratio of the primer sequence shown in SEQ ID NO.11 and the probe sequence shown in SEQ ID NO.12 is 1:10-1:2.

[0012] Fifthly, the present invention provides the use of a nucleic acid composition for multiplex PCR detection of Treponema pallidum and herpes simplex virus, or a nucleic acid composition for multiplex PCR detection of monkeypox virus, Treponema pallidum and herpes simplex virus, or the above-mentioned PCR premix in the preparation of multiplex PCR detection products.

[0013] The present invention has the following beneficial effects: This invention designs primers and probes targeting conserved regions of the genes of monkeypox virus, Treponema pallidum, and herpes simplex virus types I and II. Through in-depth analysis of the genomic sequences of each pathogen, the following targets were identified: F3L gene as the detection target for monkeypox virus; DNA polymerase I (polA) gene as the amplification and detection target for Treponema pallidum; UL42, UL30, UL27, or US6 genes as the amplification and detection targets for herpes simplex virus type I; and UL42, UL30, UL27, or US5 genes as the amplification and detection targets for herpes simplex virus type II. Primers and probes were designed from these highly conserved regions. The optimal primer-probe combination was determined through Ct value screening and melting curve shape analysis. To achieve the detection of four pathogen targets plus one internal control in the same reaction system using a fluorescence PCR instrument with four different fluorescence channels, this invention sets the fluorescent reporter groups of the two probes used to detect herpesvirus type I and herpesvirus type II to be identical or have similar spectral characteristics, and sets the fluorescent quencher groups of the two probes used to detect herpesvirus type I and herpesvirus type II to be identical or have similar spectral characteristics. By modifying the nucleic acid to increase the Tm value of the probes, the two probes for herpesvirus type I and herpesvirus type II exhibit significantly different melting curve peaks under the condition of identical groups or similar spectral characteristics. This allows for the detection of different melting curve peaks in a single fluorescence channel, and the typing of herpesvirus type I and herpesvirus type II in a single fluorescence channel.

[0014] The multiplex PCR nucleic acid detection composition provided by this invention can overcome the limitation of the number of fluorescence channels, enabling the detection of 5 targets (4 pathogens plus 1 internal control) on a 4-channel fluorescence PCR instrument, or more than 5; similarly, it can enable the detection of more than 6 targets on a 6-channel fluorescence PCR instrument.

[0015] Furthermore, the multiplex PCR detection nucleic acid composition and its application provided by this invention can simultaneously meet the needs of both subtype differentiation and non-subtype differentiation. This invention uses the same fluorescence channel for different subtypes of the same pathogen. If subtype differentiation is not required, the presence or absence of the pathogen can be detected by amplification curves. If further clarification of the pathogen's subtype is needed, melting curve analysis can be used to determine the specific subtype based on the temperature (Tm value) of the melting curve peak, without requiring additional detection. This invention offers the technical advantages of flexible application and wide applicability. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a melting curve diagram of HSV1-1F / HSV1-1R / HSV1-1P1 in Example 5; Figure 2 This is a melting curve diagram of HSV1-7F / HSV1-7R / HSV1-7P1 in Example 5; Figure 3 This is a melting curve diagram of HSV2-1F / HSV2-1R / HSV2-1P1 in Example 5; Figure 4 This is a melting curve diagram of HSV2-7F / HSV2-7R / HSV2-7P1 in Example 5; Figure 5 This is a melting curve of the herpes simplex virus type I probe in Example 6 before truncation. Figure 6 This is one of the melting curves of the truncated herpes simplex virus type I probe in Example 6; Figure 7 The second melting curve of the truncated herpes simplex virus type I probe in Example 6; Figure 8 This is the melting curve of the herpes simplex virus type II probe in Example 6 before LNA modification; Figure 9 The melting curve of the herpes simplex virus type II probe modified with LNA in Example 6 is shown. Figure 10 This is one of the experimental results graphs from the multiplex detection system for four pathogens established and optimized in Example 7; Figure 11The second figure shows the experimental results of the multiplex detection system for four pathogens established and optimized in Example 7. Figure 12 Figure 3 shows the experimental results of the multiplex detection system for four pathogens established and optimized in Example 7. Detailed Implementation

[0018] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0019] Locked nucleic acids (LNAs): A special type of XNA with a methylene "bridge" between the 2'-oxygen and 4'-carbon of its ribose, which "locks" it in a rigid conformation and has an extremely strong binding ability to complementary strands.

[0020] The detection product provided by this invention can detect samples including, but not limited to, clinical samples, cultures, swabs, and environmental samples. Clinical samples include, but are not limited to, scrapings from genital ulcer lesions, skin lesion samples, and human or animal tissues.

[0021] Environmental samples were selected from soil samples, ward samples, water samples, soil-water mixture samples, air samples, etc.

[0022] Human or animal tissues include, but are not limited to: brain, eyes, central nervous system, lymph nodes, liver and heart, etc., and body fluids include, but are not limited to: blood, serum, plasma, intracellular fluid, interstitial fluid, such as lymph and cerebrospinal fluid.

[0023] In real-time quantitative PCR, when double-stranded DNA is heated, the hydrogen bonds between its complementary bases gradually break, causing the double helix to separate into two single strands. This process is called DNA "melting." The temperature at which half of the total DNA double helix structure is degraded is called the melting temperature (Tm). Different DNA sequences have different GC content and distribution, resulting in different Tm values. As the temperature increases, the fluorescence intensity of the DNA changes. By monitoring this change, the resulting graph is the DNA melting curve.

[0024] The melting curve in this invention can detect differences in individual bases, and is low-cost, high-throughput, fast, accurate, and not limited by detection sites, achieving true closed-tube operation. Its principle is to obtain a large number of target sequences through PCR amplification, and then directly run a melting program on them. During the temperature rise, the double-stranded DNA gradually unwinds, and the dye embedded in the DNA double strand also falls off, weakening the fluorescence signal until the double-stranded DNA is completely unwinded, at which point the fluorescence signal weakens to its lowest level. DNA sequence analysis is performed by monitoring this process. Therefore, by detecting changes in the fluorescence intensity of fluorescent groups or saturated fluorescent dyes in the amplicon, a characteristic melting curve is obtained. Finally, the position and morphological changes of the melting curve are used to determine the nature of the detected template, enabling the differentiation of different pathogens.

[0025] In a first aspect, the present invention provides a nucleic acid composition for multiplex PCR detection of Treponema pallidum and herpes simplex virus, comprising: The first nucleic acid combination for detecting Treponema pallidum, as shown in SEQ ID NO.1-3, the third nucleic acid combination for detecting herpes simplex virus type I, as shown in SEQ ID NO.7-9, and the fourth nucleic acid combination for detecting herpes simplex virus type II, as shown in SEQ ID NO.10-12, wherein the nucleotides in the sequence shown in SEQ ID NO.12 are not locked nucleic acid modified or at least one nucleotide is locked nucleic acid modified; The sequences shown in SEQ ID NO.3, SEQ ID NO.9, and SEQ ID NO.12 have a fluorescent reporter group at the 5' end and a fluorescent quencher group at the 3' end; The sequence shown in SEQ ID NO. 9 has the same or similar spectral characteristics at its 5' end as the sequence shown in SEQ ID NO. 12, and the sequence shown in SEQ ID NO. 9 has the same or similar spectral characteristics at its 5' end as the sequence shown in SEQ ID NO. 12.

[0026] In a preferred embodiment of the present invention, the sequence shown in SEQ ID NO.12 has nucleotides 1-10 with locked nucleic acid modifications.

[0027] In a preferred embodiment of the present invention, the sequence shown in SEQ ID NO.12 has a locked nucleic acid modification at the 11th nucleotide starting from the 5' position.

[0028] Secondly, the present invention provides a nucleic acid composition for multiplex PCR detection of monkeypox virus, Treponema pallidum, and herpes simplex virus, comprising: The first nucleic acid combination for detecting Treponema pallidum, as shown in SEQ ID NO. 1-3; the second nucleic acid combination for detecting monkeypox virus, as shown in SEQ ID NO. 4-6; the third nucleic acid combination for detecting herpes simplex virus type I, as shown in SEQ ID NO. 7-9; and the fourth nucleic acid combination for detecting herpes simplex virus type II, as shown in SEQ ID NO. 10-12, wherein the nucleotides in the sequence shown in SEQ ID NO. 12 are not locked nucleic acid modified or have at least one locked nucleic acid modified.

[0029]

[0030] The sequences shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, and SEQ ID NO.12 have a fluorescent reporter group at their 5' end and a fluorescent quencher group at their 3' end. The sequence shown in SEQ ID NO.9 has the same or similar fluorescent reporter group at its 5' end as the sequence shown in SEQ ID NO.12, and the sequence shown in SEQ ID NO.9 has the same or similar fluorescent quencher group at its 5' end as the sequence shown in SEQ ID NO.12.

[0031] This invention utilizes a four-channel fluorescent PCR instrument to simultaneously detect four pathogens: Treponema pallidum, herpes simplex virus type I, herpes simplex virus type II, and monkeypox virus, along with an internal control. Considering that herpes simplex virus type I and herpes simplex virus type II share approximately 45% to 60% nucleic acid sequence homology, they have similar sequences in certain regions of their genomes, but also exhibit significant differences. This invention employs a unique design, selecting specific regions on the genomes of herpes simplex virus type I and herpes simplex virus type II to design primers and probes, labeling the probes with the same fluorescent group but with different melting temperatures (Tm). In the fluorescent PCR reaction, regardless of the presence of nucleic acid templates from either herpes simplex virus type I or herpes simplex virus type II, fluorescence signals and amplification curves are generated in the same channel. After PCR amplification is completed, melting curve analysis is performed. Since the probes of herpes simplex virus type I and herpes simplex virus type II are different, they have characteristic melting curves and specific melting temperatures. Therefore, herpes simplex virus type I and herpes simplex virus type II can be distinguished by melting temperature.

[0032] Different fluorescent groups were used for Treponema pallidum, monkeypox virus, and internal reference gene, respectively. The same fluorescent group was used for herpes simplex virus type I and herpes simplex virus type II, but they were distinguished by different melting temperatures. By combining the analysis of fluorescence channels and melting temperatures, a fluorescence PCR instrument with 4 different fluorescence channels was used to detect 4 pathogens plus 1 internal reference gene in the same reaction.

[0033] In a preferred embodiment of the present invention, the sequence shown in SEQ ID NO. 12 has locked nucleic acid (LNA) modifications at positions 1-10; for example, the sequence shown in SEQ ID NO. 12 has locked nucleic acid (LNA) modifications at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0034] The nucleotides modified by locked nucleic acids can be continuous or discontinuous, and the modification can be located at the 5' end, 3' end, or in the middle.

[0035] In a preferred embodiment of the present invention, the sequence shown in SEQ ID NO. 12 has a locked nucleic acid modification at position 11 starting from 5'. The modified sequence has an extremely high Tm value, causing the two probes for herpesvirus type I and herpesvirus type II to have significantly different melting curve peaks under conditions of identical functional groups or similar spectral characteristics. This enables the detection of different melting curve peaks in a single fluorescence channel, and the typing of herpesvirus type I and herpesvirus type II in a single fluorescence channel.

[0036] The fluorescent reporter group is selected from any one of 5-FAM, 6-FAM, HEX, TET, VIC, JOE, Cy3, Cy3.5, NED, TAMRA, ROX, TexasRed, Cy5, Cy5.5 and Quasar670, and the fluorescent quencher group is selected from any one of TAMRA, BHQ1, BHQ2, BHQ3, MGB and QSY.

[0037] Thirdly, the present invention provides a nucleic acid composition for multiplex PCR detection of Treponema pallidum and herpes simplex virus or a multiplex PCR detection product for monkeypox virus, Treponema pallidum and herpes simplex virus, comprising the aforementioned nucleic acid composition for multiplex PCR detection of monkeypox virus, Treponema pallidum and herpes simplex virus.

[0038] In a preferred embodiment of the present invention, the multiplex PCR detection product is selected from reagents, kits, chips, or detectors.

[0039] In a preferred embodiment of the present invention, the multiplex PCR detection product further includes at least one of the following components: internal reference primer, internal reference probe, PCR buffer, dNTPs, DNA polymerase, positive control, negative control, and water.

[0040] The internal reference primers and probes target internal reference genes selected from at least one of GAPDH, β-Actin, U6, HMBS, B2M, TUBB, SDHA, 18S rRNA, ACTB, RPL4, PPIA, HPRT1, YWHAZ, RPP30, and ERG.

[0041] In a preferred embodiment of the present invention, the nucleotide sequence of the internal reference primer probe is shown in SEQ ID NO.13-15.

[0042] Fourthly, the present invention provides a PCR premix comprising the above-mentioned multiplex PCR detection nucleic acid composition for Treponema pallidum and herpes simplex virus or a multiplex PCR detection nucleic acid composition for monkeypox virus, Treponema pallidum and herpes simplex virus; wherein the final concentration ratio of the primer sequences shown in SEQ ID NO. 1-2 to the probe sequences shown in SEQ ID NO. 3 in the PCR premix is ​​1:1-10:1. In the PCR premix, the final concentration ratio of the primer sequences shown in SEQ ID NO. 1-2 to the probe sequence shown in SEQ ID NO. 3 is 1:1-10:1; the final concentration ratio of the primer sequences shown in SEQ ID NO. 4-5 to the probe sequence shown in SEQ ID NO. 6 is 1:1-10:1; the final concentration ratio of the primer sequences shown in SEQ ID NO. 7 and SEQ ID NO. 8 is 2:1-10:1; and the final concentration ratio of the primer sequence shown in SEQ ID NO. 8 to the probe sequence shown in SEQ ID NO. 9 is 1:10-1:2. The final concentration ratio of the primer sequences shown in SEQ ID NO.10 and SEQ ID NO.11 is 2:1-10:1; the final concentration ratio of the primer sequence shown in SEQ ID NO.11 and the probe sequence shown in SEQ ID NO.12 is 1:10-1:2.

[0043] In one embodiment, the final concentration ratio of the primer sequence shown in SEQ ID NO. 1-2 to the probe sequence shown in SEQ ID NO. 3 in the PCR premix is ​​1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1. In one embodiment, the final concentration ratio of the primer sequences shown in SEQ ID NO. 1-2 to the probe sequence shown in SEQ ID NO. 3 in the PCR premix is ​​1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1; the final concentration ratio of the primer sequences shown in SEQ ID NO. 4-5 to the probe sequence shown in SEQ ID NO. 6 is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1; the final concentration ratio of the primer sequences shown in SEQ ID NO. 7 to SEQ ID NO. 8 is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1; the final concentration ratio of the primer sequences shown in SEQ ID NO. 8 to SEQ ID NO. 3 is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1; the final concentration ratio of the primer sequences shown in SEQ ID NO. 7 to SEQ ID NO. 8 is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1; the final concentration ratio of the primer sequences shown in SEQ ID NO. 8 to SEQ ID NO. 3 is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. The final concentration ratio of the probe sequence shown in IDNO.9 is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10; The final concentration ratio of the primer sequences shown in SEQ ID NO.10 and SEQ ID NO.11 is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1; the final concentration ratio of the primer sequence shown in SEQ ID NO.11 to the probe sequence shown in SEQ ID NO.12 is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0044] By using an asymmetric PCR system, one strand of the double-stranded DNA of herpes simplex virus type I and type II can be preferentially amplified to obtain single-stranded DNA.

[0045] Under the above reaction system, it has high detection specificity, sensitivity and melting curve, and can accurately classify herpesvirus type I and type II.

[0046] In a preferred embodiment of the present invention, the PCR premix also includes an internal reference primer and an internal reference probe, with a final concentration of the internal reference primer to the internal reference probe being 1:1 to 10:1. For example, it is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0047] Fifthly, the present invention provides the use of a nucleic acid composition for multiplex PCR detection of Treponema pallidum and herpes simplex virus, or a nucleic acid composition for multiplex PCR detection of monkeypox virus, Treponema pallidum and herpes simplex virus, or the above-mentioned PCR premix in the preparation of multiplex PCR detection products.

[0048] In a preferred embodiment of the present invention, the application includes the following methods: Preparation of the PCR reaction system: nucleic acid of the sample to be tested, PCR buffer, DNA polymerase, dNTPs, primer sequences shown in SEQ ID NO. 1-2 (100-800 nM), probe sequence shown in SEQ ID NO. 3 (50-600 nM); primer sequences shown in SEQ ID NO. 4-5 (100-800 nM), probe sequence shown in SEQ ID NO. 6 (50-600 nM); primer sequences shown in SEQ ID NO. 7 (100-800 nM), primer sequences shown in SEQ ID NO. 8 (30-500 nM), probe sequence shown in SEQ ID NO. 9 (50-600 nM), primer sequences shown in SEQ ID NO. 10 (50-600 nM), primer sequences shown in SEQ ID NO. 11 (30-500 nM), probe sequence shown in SEQ ID NO. 12 (50-600 nM), and dNTPs of the sample to be tested. Primer sequences shown in ID NO. 13-14 and probe sequences shown in SEQ ID NO. 15 (30-500 nM).

[0049] Preferably, the PCR reaction system is prepared as follows: nucleic acid of the sample to be tested, PCR buffer, DNA polymerase, dNTPs, 400 nM of the primer sequences shown in SEQ ID NO. 1-2, 200 nM of the probe sequence shown in SEQ ID NO. 3; 400 nM of the primer sequences shown in SEQ ID NO. 4-5, 200 nM of the probe sequence shown in SEQ ID NO. 6; 360 nM of the primer sequence shown in SEQ ID NO. 7, 60 nM of the primer sequence shown in SEQ ID NO. 8, 200 nM of the probe sequence shown in SEQ ID NO. 9, 360 nM of the primer sequence shown in SEQ ID NO. 10, 60 nM of the primer sequence shown in SEQ ID NO. 11, 200 nM of the probe sequence shown in SEQ ID NO. 12, 160 nM of the primer sequences shown in SEQ ID NO. 13-14, and 80 nM of the probe sequence shown in SEQ ID NO. 15. Then, the amplification reaction was carried out under the following reaction procedure: At 95-100℃, 2 min; 95℃, 10 s; 56-62℃, 30 s; repeat 40-45 times.

[0050] DNA polymerase, preferably a hot-start DNA polymerase, such as Tth DNA polymerase, Taq DNA polymerase, etc.

[0051] The PCR buffer includes buffer system reagents, including but not limited to the PB series and Tris series. The PCR buffer includes lyophilization protectants, such as at least one of mannitol, trehalose, dextran, gelatin, hydrogenated maltose, and sucrose.

[0052] The PCR buffer also contains anti-PCR inhibitor factors, such as spermidine, trehalose, betaine, etc., making the nucleic acid composition provided by this invention more suitable for the detection of complex animal clinical samples.

[0053] The detection products (such as kits) provided by the present invention may optionally include any reagents and / or consumables acceptable in the art for PCR reactions or for preparing PCR reaction systems. Specific embodiments may include, but are not limited to, one or more of blank controls, calibrators, and PCR reaction containers.

[0054] In one feasible implementation, a melting procedure can be performed: a gradient temperature increase from 40℃ to 90℃, followed by melting curve analysis.

[0055] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0056] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0057] Example 1 This embodiment involves the screening of Treponema pallidum primers and probes. The sequence of Treponema pallidum (TP) was obtained from the National Center for Biotechnology Information (NCBI) database. Using PrimerPremier 3 software, highly conserved regions of the gene were selected for primer and probe design. Through in-depth analysis of the Treponema pallidum genome sequence, the DNA polymerase I (polA) gene (GenBank Gene ID: 93875899) was identified as the target for amplification and testing, and its conserved regions were selected. Based on the principles and methods of quantitative real-time PCR (qPCR), primers and probes were carefully designed, and the primer and probe sequences were synthesized by a professional supplier.

[0058] Three sets of primers and probes were designed and synthesized targeting the polA gene of Treponema pallidum. Reaction systems were prepared using three different primer-probe combinations (TP-1F / TP-1R / TP-1P, TP-2F / TP-2R / TP-2P, and TP-3F / TP-3R / TP-3P) to detect DNA from two Treponema pallidum-positive samples and one negative sample. Each sample was tested twice. The optimal primers and probes were selected based on the amplification curve shape and Ct value of each primer-probe combination.

[0059] Reaction system:

[0060] Reaction procedure:

[0061] Experimental results:

[0062] Experimental results show that the TP-3F / TP-3R / TP-3P primer-probe combination exhibits good performance. All primer-probe sequences are as follows:

[0063] In this embodiment, TP-3P is labeled as 5'-Cy5,3'-BHQ3, and the change of the labeling group does not affect the protection range of the sequence.

[0064] Example 2 This embodiment involves screening primers and probes for monkeypox.

[0065] The sequence of monkeypox virus (MPXV) was obtained from the National Center for Biotechnology Information (NCBI) database. Using PrimerPremier 3 software, highly conserved regions of the gene were selected for primer and probe design. Through in-depth analysis of the monkeypox virus genome sequence, the F3L gene (GenBank Gene ID: 928998) was identified as the target for amplification and testing, and its conserved regions were selected. Based on the principles and methods of quantitative real-time PCR (qPCR), primers and probes were carefully designed, and the primer and probe sequences were synthesized by a professional supplier.

[0066] Three sets of primers and probes were designed and synthesized targeting the F3L gene of monkeypox virus. Different combinations of these three primers and probes were then used. Prepare reaction systems for (MPXV-1F / MPXV-1R / MPXV-1P, MPXV-2F / MPXV-2R / MPXV-2P, and MPXV-3F / MPXV-3R / MPXV-3P). DNA from two monkeypox-positive samples and one monkeypox-negative sample was tested, with each sample tested twice. The optimal primers and probes were selected based on the amplification curve shape and Ct value of different primers and probes. The reaction system and procedure were the same as in Example 1.

[0067] The experimental results are as follows:

[0068] Experimental results show that the MPXV-2P primer-probe combination performs well. All primer-probe sequences are as follows:

[0069] In this embodiment, MPXV-2P is labeled as 5'6-FAM, 3'BHQ-1. Changing the labeling group does not affect the protection range of the sequence.

[0070] Example 3 The sequence of Herpes simplex virus type 1 (HSV-1) was obtained from the National Center for Biotechnology Information (NCBI) database. Using Primer Premier 3 software, highly conserved regions of the gene were selected for primer and probe design. Through in-depth analysis of the HSV-1 genome sequence, the UL42 gene (GenBank Gene ID: 24271471), UL30 gene (GenBank Gene ID: 2703462), UL27 gene (GenBank Gene ID: 24271469), or US6 gene (GenBank Gene ID: 2703444) were identified as amplification and detection targets, and their conserved regions were selected. Primers were carefully designed based on the principles and methods of quantitative real-time PCR, and primer sequences were synthesized by a professional supplier.

[0071] Eight primer-probe combinations were designed and synthesized targeting highly conserved regions of the herpes simplex virus type I genome. The fluorescent dye EvaGreen was used for primer screening experiments. Reaction systems were prepared using these different primer combinations to detect nucleic acid samples of varying concentrations extracted from HSV1 cultures, with each sample tested twice. During the screening process, a preliminary assessment was first made based on the melting curve shape of each primer, followed by further analysis using the Ct values ​​of the amplification curves to preliminarily determine the optimal primer combinations.

[0072] Reaction system:

[0073] Reaction procedure:

[0074] Note: * indicates that fluorescence was collected in this step.

[0075] Experimental results:

[0076] Based on the experimental results and the specificity of the melting curves (results not shown), two primer sets (HSV1-1F / HSV1-1R and HSV1-7F / HSV1-7R) were selected for subsequent probe screening. The sequences of all primer combinations are as follows:

[0077] Example 4 Screening of primers for herpes simplex virus type II The sequence of Herpes simplex virus type 2 (HSV-2) was obtained from the National Center for Biotechnology Information (NCBI) database. Primer and probe design was performed using Primer Premier 3 software to select highly conserved regions of the gene. Through in-depth analysis of the HSV-2 genome sequence, the UL42 gene (GenBank Gene ID: 1487329), UL30 gene (GenBank Gene ID: 1487316), UL27 gene (GenBank Gene ID: 1487312), and US5 gene (GenBank Gene ID: 1487357) were identified as amplification and detection targets, and their conserved regions were selected. Primers were carefully designed based on the principles and methods of quantitative real-time PCR, and primer sequences were synthesized by a professional supplier.

[0078] Eight primer-probe combinations were designed and synthesized targeting highly conserved regions of the herpes simplex virus type II genome. The fluorescent dye EvaGreen was used for primer screening experiments. Reaction systems were prepared using these different primer combinations to detect nucleic acid samples of varying concentrations extracted from HSV2 cultures, with each sample tested twice. During the screening process, a preliminary assessment was first made based on the melting curve shape of each primer, followed by further analysis using the Ct values ​​of the amplification curves to preliminarily determine the optimal primer combinations.

[0079] The reaction system and procedure are the same as in Example 3.

[0080] Experimental results:

[0081] Based on the experimental results and the specificity of the melting curves (results not shown), two primer sets (HSV2-1F / HSV2-1R and HSV2-7F / HSV2-7R) were selected for subsequent probe screening. The sequences of all primer combinations are as follows:

[0082] Example 5 Screening of herpes simplex virus type I and type II probes Based on the primer sequences obtained from the aforementioned screening, probes were designed within the corresponding primer amplification regions using the genome sequences of herpes simplex virus type I and type II, respectively.

[0083] Based on the two primer pairs selected from both herpes simplex virus type I and herpes simplex virus type II, one probe was designed for each primer pair, resulting in a total of four primer-probe combinations. Asymmetric PCR reaction systems were prepared using these primer-probe combinations to detect nucleic acids extracted from HSV1 or HSV2 cultures or negative samples, with each sample tested twice. During the screening process, a preliminary assessment was first made based on the melting curve shape of each primer, followed by further analysis using the Ct value of the amplification curve to ultimately determine the optimal primer-probe combination.

[0084] Reaction system:

[0085] Experimental results:

[0086]

[0087] Melting curve as shown Figure 1 , Figure 2 , Figure 3 , Figure 4 As shown in the experimental results, we can see that: For the detection of herpes simplex virus type 1 (HSV1), the primer-probe combination HSV1-7F / HSV1-7R / HSV1-7P exhibits a low Ct value and good peak shape, with a clear single peak in the melting curve, indicating that the amplification product is mainly a single, specific DNA fragment. Therefore, HSV1-7F / HSV1-7R / HSV1-7P1 is the preferred primer-probe combination for HSV1.

[0088] For the detection of herpes simplex virus type 2 (HSV2), the primer-probe combination HSV2-1F / HSV2-1R / HSV2-1P exhibits a low Ct value and good peak shape, with a clear single peak in the melting curve, indicating high specificity of its amplified products. Therefore, HSV2-1F / HSV2-1R / HSV2-1P1 is the preferred primer-probe combination for HSV2.

[0089] However, the average Tm value of the melting curve peaks obtained by HSV1-7F / HSV1-7R / HSV1-7P1 was 65.43℃, while the average Tm value of the melting curve peaks obtained by HSV2-1F / HSV2-1R / HSV2-1P1 was 69.23℃, with a difference of less than 5℃. This means that when detecting two pathogens in the same channel, it may be difficult to effectively distinguish their amplification products based on Tm values. Therefore, when it is necessary to simultaneously detect HSV1 and HSV2, further optimization of the two primer pairs is required to ensure accurate differentiation of HSV1 and HSV2 amplification products.

[0090] The final selected primer and probe sequences are as follows:

[0091] In this embodiment, HSV1-7P1 and HSV2-1P1 are labeled as 5'ROX, 3'BHQ-2. The change of the labeling group does not affect the protection range of the sequence.

[0092] Example 6 Optimization of probes for herpes simplex virus type I and type II The optimal probe combination obtained in Example 4 has probe Tm values ​​of approximately 65 degrees and 69 degrees for HSV1 and HSV2, respectively. To further increase the difference in Tm values ​​between these two probes (≥5°C) and thus enhance differentiation and identification, the probe was further optimized.

[0093] HSV1 probes lower the Tm value by reducing bases, while HSV2 probes increase the Tm value by adding locked nucleic acids. All probes were evaluated using Tm prediction software (https: / / geneglobe.qiagen.com / us / tools / tm-prediction). We used these probes and primer combinations to perform asymmetric PCR detection on nucleic acids extracted from HSV1, HSV2 cultures, and negative samples, with each sample tested twice. The optimal primer-probe combination was determined by analyzing melting curves and Ct values.

[0094] The specific probe sequences optimized in the following order are as follows:

[0095] Prepare five sets of asymmetric PCR reaction systems with primers and probes according to the formula in the table below. Except for the primers and probes, all other materials are the same. Add 5.0 μL of nucleic acid sample to a 20 μL reaction system for reaction. Detect each nucleic acid sample in parallel twice.

[0096]

[0097] Use the same reaction procedure as in Example 3.

[0098] Experimental results:

[0099]

[0100]

[0101] Melting curve reference Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 As shown: Based on the statistical results of the average Tm difference, five probe combinations that initially met the requirements were obtained (Tm difference between HSV1 and HSV2 probes ≥ 5℃). Further evaluation of the detection performance of HSV1-7P1 and its truncated probe sequences revealed that the probe HSV1-7P1-m2, which was truncated by two bases, caused a delay in the Ct value, indicating a decrease in its amplification efficiency, and was therefore excluded. Among the remaining two qualified probe combinations (HSV2-1P1-m1 + HSV1-7P1 and HSV2-1P1-m1 + HSV1-7P1-m1), based on the principle of maximizing the Tm difference, HSV2-1P1-m1 + HSV1-7P1-m1 was finally selected as the optimal probe combination.

[0102] Example 7 This embodiment establishes and optimizes a multiplex detection system for four pathogens. Based on the screening results of primers and probes for syphilis, monkeypox, herpes simplex virus type I, and herpes simplex virus type II, and using the B2M gene as an internal control, we formulated a multiplex reaction system using primers and probes for the four pathogens and the internal control. Nucleic acid samples extracted from HSV1 cultures, HSV2 cultures, mixed positive samples, syphilis clinical samples, monkeypox clinical samples, and negative samples were tested, with each sample tested twice.

[0103] Prepare asymmetric PCR reaction systems for primers and probes according to the formulas in the table below. Except for the primers and probes, all other materials are the same. Add 5 μL of nucleic acid sample to a 20 μL reaction system for reaction. Detect each nucleic acid sample in parallel twice.

[0104] Reaction procedure:

[0105] Note: * indicates that fluorescence was collected in this step. Reaction system:

[0106] Primer sequences:

[0107] The fluorescent probe labeling combination used in this combination can be modified without affecting the scope of protection of this invention.

[0108] Experimental samples:

[0109] Experimental results:

[0110] Experimental results refer to Figure 10 , Figure 11 , Figure 12As shown, primers and probes for Treponema pallidum, monkeypox virus (MPXV), herpes simplex virus type 1 (HSV1), herpes simplex virus type 2 (HSV2), and an internal control (IC) were formulated into a multiplex real-time fluorescent PCR system for testing. The results showed that all targets could be stably detected. HSV1 and HSV2 exhibited good peak shapes in both amplification and melting curves, showing a single, sharp melting peak, indicating high amplification specificity. Furthermore, although both were detected in the same fluorescent channel, their melting temperatures (Tm values) differed by more than 5°C, thus allowing for clear differentiation of their amplification products through melting curve analysis.

[0111] Example 8 This embodiment performs sensitivity testing on a multiplex reaction system for four pathogens. High-concentration HSV1 positive samples, HSV2 positive samples, TP positive samples, and MPXV positive samples were serially diluted 10-fold with negative samples to prepare 10... 0 10 -1 10 -2 10 -3 10 -4 Samples at different dilutions were prepared. Following the protocol in Implementation 7, primers and probes for the four pathogens and the internal control were combined to form a multiplex reaction system. Then, PCR detection was performed on samples at each dilution, and the lowest dilution that could be detected was recorded.

[0112] The test results are as follows:

[0113] Experimental results showed that HSV1 positive samples and MPXV were present in 10 samples. -3 It can still be detected after dilution, 10 -4 Diluted negative, sensitivity 10 for all. -3 HSV2 positive samples and TP positive samples in 10 -5 It can still be detected after dilution, 10 -6 Diluted negative, sensitivity 10 for all. -5 The Tm values ​​of each gradient probe were stable, and the linear equation for Ct concentration had R² ≥ 0.997, indicating a good linear relationship, which confirms the reliability and stability of the method.

[0114] Example 9 This embodiment validates clinical samples using a multiplex reaction system for four pathogens. To verify the effectiveness of the kit of the present invention in detecting clinical samples, pathogen samples from the skin surface of patients suspected of syphilis, herpes simplex virus, or monkeypox infection, such as chancres, ulcers, and rashes, were collected. Nucleic acid was extracted, or the samples were treated with a nucleic acid release agent, and then detected using the kit of Example 7 of the present invention. The commercially available kits "Treponema pallidum and herpes simplex virus type I / II typing nucleic acid detection kit (fluorescent PCR method)" and "Monkeypox virus nucleic acid detection kit (fluorescent PCR method)" from Shanghai GeneoBio Corporation were used as reference reagents.

[0115] The test results for 67 samples are as follows.

[0116] The results showed that the reagents used in this invention produced detection results that were highly consistent with those of commercially available products.

[0117] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nucleic acid composition for multiplex PCR detection of Treponema pallidum and herpes simplex virus, characterized in that, It includes: The first nucleic acid combination for detecting Treponema pallidum, as shown in SEQ ID NO.1-3, the third nucleic acid combination for detecting herpes simplex virus type I, as shown in SEQ ID NO.7-9, and the fourth nucleic acid combination for detecting herpes simplex virus type II, as shown in SEQ ID NO.10-12, wherein the nucleotides in the sequence shown in SEQ ID NO.12 are not locked nucleic acid modified or at least one nucleotide is locked nucleic acid modified; The sequences shown in SEQ ID NO.3, SEQ ID NO.9, and SEQ ID NO.12 have a fluorescent reporter group at the 5' end and a fluorescent quencher group at the 3' end; The sequence shown in SEQ ID NO. 9 has the same or similar spectral characteristics at its 5' end as the sequence shown in SEQ ID NO. 12, and the sequence shown in SEQ ID NO. 9 has the same or similar spectral characteristics at its 5' end as the sequence shown in SEQ ID NO.

12.

2. The nucleic acid composition for multiplex PCR detection of Treponema pallidum and herpes simplex virus according to claim 1, characterized in that, The sequence shown in SEQ ID NO.12 has locked nucleic acid modifications at positions 1-10.

3. The nucleic acid composition for multiplex PCR detection of Treponema pallidum and herpes simplex virus according to claim 1, characterized in that, The sequence shown in SEQ ID NO.12 has a locked nucleic acid modification at position 11 starting from 5'.

4. A nucleic acid composition for multiplex PCR detection of monkeypox virus, Treponema pallidum, and herpes simplex virus, characterized in that, It includes: The first nucleic acid combination for detecting Treponema pallidum, as shown in SEQ ID NO. 1-3; the second nucleic acid combination for detecting monkeypox virus, as shown in SEQ ID NO. 4-6; the third nucleic acid combination for detecting herpes simplex virus type I, as shown in SEQ ID NO. 7-9; and the fourth nucleic acid combination for detecting herpes simplex virus type II, as shown in SEQ ID NO. 10-12, wherein the nucleotides in the sequence shown in SEQ ID NO. 12 are not locked nucleic acid modified or at least one nucleotide is locked nucleic acid modified; The sequences shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, and SEQ ID NO.12 have a fluorescent reporter group at the 5' end and a fluorescent quencher group at the 3' end; The sequence shown in SEQ ID NO. 9 has the same or similar spectral characteristics at its 5' end as the sequence shown in SEQ ID NO. 12, and the sequence shown in SEQ ID NO. 9 has the same or similar spectral characteristics at its 5' end as the sequence shown in SEQ ID NO.

12.

5. The nucleic acid composition for multiplex PCR detection of monkeypox virus, Treponema pallidum, and herpes simplex virus according to claim 4, characterized in that, The sequence shown in SEQ ID NO.12 has locked nucleic acid modifications at positions 1-10.

6. The nucleic acid composition for multiplex PCR detection of monkeypox virus, Treponema pallidum, and herpes simplex virus according to claim 4, characterized in that, The sequence shown in SEQ ID NO.12 has a locked nucleic acid modification at position 11 starting from 5'.

7. A multiplex PCR detection product, characterized in that, It includes the nucleic acid composition for multiplex PCR detection of Treponema pallidum and herpes simplex virus as described in any one of claims 1-3 or the nucleic acid composition for multiplex PCR detection of monkeypox virus, Treponema pallidum and herpes simplex virus as described in any one of claims 4-6; Preferably, the multiplex PCR detection product is selected from reagents, kits, chips, or detectors; Preferably, the multiplex PCR detection product further includes at least one of the following components: internal reference primer, internal reference probe, PCR buffer, dNTPs, DNA polymerase, positive control, negative control, and water; Preferably, the nucleotide sequence of the internal reference primer probe is shown in SEQ ID NO.13-15.

8. A PCR premix, characterized in that, It includes the nucleic acid composition for multiplex PCR detection of Treponema pallidum and herpes simplex virus as described in any one of claims 1-3 or the nucleic acid composition for multiplex PCR detection of monkeypox virus, Treponema pallidum and herpes simplex virus as described in any one of claims 4-6; the final concentration ratio of the primer sequence shown in SEQ ID NO. 1-2 to the probe sequence shown in SEQ ID NO. 3 in the PCR premix is ​​1:1-10:1; In the PCR premix, the final concentration ratio of the primer sequences shown in SEQ ID NO. 1-2 to the probe sequence shown in SEQ ID NO. 3 is 1:1-10:1; the final concentration ratio of the primer sequences shown in SEQ ID NO. 4-5 to the probe sequence shown in SEQ ID NO. 6 is 1:1-10:1; the final concentration ratio of the primer sequences shown in SEQ ID NO. 7 and SEQ ID NO. 8 is 2:1-10:1; and the final concentration ratio of the primer sequence shown in SEQ ID NO. 8 to the probe sequence shown in SEQ ID NO. 9 is 1:10-1:

2. The final concentration ratio of the primer sequences shown in SEQ ID NO.10 and SEQ ID NO.11 is 2:1-10:1; the final concentration ratio of the primer sequence shown in SEQ ID NO.11 and the probe sequence shown in SEQ ID NO.12 is 1:10-1:

2. Preferably, the PCR premix further includes an internal reference primer and an internal reference probe, wherein the final concentration of the internal reference primer to the internal reference probe is 1:1-10:

1.

9. The use of the nucleic acid composition for multiplex PCR detection of Treponema pallidum and herpes simplex virus as described in any one of claims 1-3, the nucleic acid composition for multiplex PCR detection of monkeypox virus, Treponema pallidum and herpes simplex virus as described in any one of claims 4-6, or the PCR premix as described in claim 8 in the preparation of multiplex PCR detection products.

10. The application according to claim 9, characterized in that, The application includes the following application methods: Preparation of the PCR reaction system: nucleic acid of the sample to be tested, PCR buffer, DNA polymerase, dNTPs, primer sequences shown in SEQ ID NO. 1-2 (100-800 nM), probe sequence shown in SEQ ID NO. 3 (50-600 nM); primer sequences shown in SEQ ID NO. 4-5 (100-800 nM), probe sequence shown in SEQ ID NO. 6 (50-600 nM); primer sequences shown in SEQ ID NO. 7 (100-800 nM), primer sequences shown in SEQ ID NO. 8 (30-500 nM), probe sequence shown in SEQ ID NO. 9 (50-600 nM), primer sequences shown in SEQ ID NO. 10 (50-600 nM), primer sequences shown in SEQ ID NO. 11 (30-500 nM), probe sequence shown in SEQ ID NO. 12 (50-600 nM), and probe sequence shown in SEQ ID NO. 11 (50-600 nM). Primer sequences shown in NO.13-14 and probe sequence shown in SEQ ID NO.15 (30-500 nM); Then, the amplification reaction was carried out under the following reaction procedure: At 95-100℃, 2 min; 95℃, 10 s; 56-62℃, 30 s; repeat 40-45 times. Preferably, the following melting procedure is performed: a gradient temperature increase from 40℃ to 90℃, followed by melting curve analysis.

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