A specific primer set, specific probe for simultaneously detecting and distinguishing 6 subtypes of cicovirus and application thereof

Abstract

The present application relates to the technical field of molecular biology detection, and provides a specific primer set and a specific probe for simultaneously detecting and distinguishing six subtypes of circovirus, including TTV-29 subtype, TTV-16 subtype, TTV-15 subtype, TTV-24 subtype, TTV-1 subtype and TTV-19 subtype. The specific primer set and the specific probe are used for qPCR amplification, so that the six subtypes of circovirus most commonly seen in HSCT patients can be simultaneously detected and distinguished, the detection speed is high, the specificity is strong, and the problems of low detection efficiency and insufficient typing capacity in the prior art are solved; meanwhile, the detection method is simple, the detection time is short, the cost is low, the accuracy is high, and patients are more likely to accept.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, and in particular to a specific primer set, a specific probe, and their applications for simultaneously detecting and distinguishing six circovirus subtypes. Background Technology

[0002] Anelloviridae is a group of circular single-stranded DNA viruses that are widely distributed in the human population. They are diverse in species and complex in subtypes. Existing detection methods mainly focus on: (1) total detection: using universal primers to detect all anelloviridae, but cannot distinguish between different subtypes; (2) singleton PCR detection: can detect specific subtypes, but is inefficient and time-consuming; (3) metagenomic sequencing (mNGS): although it can genotype, it is costly, requires enrichment, and has a long detection cycle, making it difficult to use routinely.

[0003] In summary, there is currently a lack of a simple method that can rapidly and specifically detect and classify the dominant subtypes of circoviruses simultaneously. Summary of the Invention

[0004] The purpose of this invention is to provide a specific primer set and specific probe for simultaneously detecting and distinguishing six circovirus subtypes. By using the specific primer set and specific probe described in this invention to detect samples, it is possible to simultaneously detect and distinguish the six most common circovirus subtypes in HSCT patients, with fast detection speed and high specificity.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a specific primer set and specific probe for simultaneously detecting and distinguishing six circovirus subtypes, including TTV-29, TTV-16, TTV-15, TTV-24, TTV-1, and TTV-19.

[0007] Preferably, the specific primer set includes the TTV-29 subtype specific primer set, the TTV-16 subtype specific primer set, the TTV-15 subtype specific primer set, the TTV-24 subtype specific primer set, the TTV-1 subtype specific primer set, and the TTV-19 subtype specific primer set.

[0008] The TTV-29 subtype-specific primer set includes a forward primer with the sequence shown in SEQ ID NO:1 and a reverse primer with the sequence shown in SEQ ID NO:2;

[0009] The TTV-16 subtype-specific primer set includes a forward primer with the sequence shown in SEQ ID NO:4 and a reverse primer with the sequence shown in SEQ ID NO:5;

[0010] The TTV-15 subtype-specific primer set includes a forward primer with the sequence shown in SEQ ID NO:7 and a reverse primer with the sequence shown in SEQ ID NO:8;

[0011] The TTV-24 subtype-specific primer set includes a forward primer with the sequence shown in SEQ ID NO:10 and a reverse primer with the sequence shown in SEQ ID NO:11;

[0012] The TTV-1 subtype-specific primer set includes a forward primer with a sequence as shown in SEQ ID NO:13 and a reverse primer with a sequence as shown in SEQ ID NO:14;

[0013] The TTV-19 subtype-specific primer set includes a forward primer with a sequence as shown in SEQ ID NO:16 and a reverse primer with a sequence as shown in SEQ ID NO:17.

[0014] Preferably, the specific probes include TTV29-P with the sequence shown in SEQ ID NO:3, TTV16-P with the sequence shown in SEQ ID NO:6, TTV15-P with the sequence shown in SEQ ID NO:9, TTV24-P with the sequence shown in SEQ ID NO:12, TTV1-P with the sequence shown in SEQ ID NO:15, and TTV19-P with the sequence shown in SEQ ID NO:18.

[0015] Preferably, the 5' end of the specific probe is labeled with a fluorescent reporter group, and the 3' end is labeled with an NFQ-MGB group; the fluorescent reporter group includes Texas Red, VIC, FAM, and Cy.

[0016] This invention also provides the application of the specific primer set and specific probe in the preparation of products that simultaneously detect and distinguish six circovirus subtypes.

[0017] Preferably, the product includes a reagent kit.

[0018] The present invention also provides a kit for simultaneously detecting and distinguishing six circovirus subtypes, including the aforementioned specific primer set and specific probe.

[0019] The present invention also provides a method for simultaneously detecting and differentiating six circovirus subtypes for purposes other than disease diagnosis and / or treatment, comprising the following steps:

[0020] (1) Extract DNA from the sample to be tested to obtain a DNA sample;

[0021] (2) The DNA sample from step (1) is amplified by qPCR using the specific primer set and specific probe, and the results are determined based on the fluorescence curve and Ct value of the amplification product.

[0022] A positive result is indicated by the appearance of an S-shaped fluorescence curve and a Ct value ≤ 38; a negative result is indicated by a Ct value > 38 or the absence of an S-shaped curve.

[0023] Preferably, the qPCR amplification system further includes an internal reference primer set and an internal reference probe, wherein the internal reference primer set consists of a forward primer with the sequence shown in SEQ ID NO.19 and a reverse primer with the sequence shown in SEQ ID NO.20.

[0024] Preferably, the internal reference probe is HPRT1-P with the sequence shown in SEQ ID NO.21.

[0025] Preferably, the internal reference probe has a VIC group attached to its 5' end and an NFQ-MGB attached to its 3' end.

[0026] Preferably, the amplification system comprises: 10 μL of 2× qPCR premix, 0.3 μL each of 0.3 μM specific forward primers, 0.3 μL each of 0.3 μM specific reverse primers, 0.3 μL of 0.3 μM internal control forward primer, 0.3 μL of 0.3 μM internal control reverse primer, 0.2 μL each of 0.10 μM specific probes, 0.2 μL of 0.10 μM internal control probe, 2 μL of template DNA, and finally, nuclease-free water to a final volume of 20 μL.

[0027] Preferably, the amplification program is as follows: pre-denaturation at 95℃ for 3 min; 95℃ for 15 s, 60℃ for 30 s, 40 cycles; 72℃ for 30 s.

[0028] By adopting the above technical solution, the present invention has the following beneficial effects:

[0029] The technical solution of this invention utilizes the specific primer set and specific probe for qPCR amplification and detection, which can simultaneously detect and differentiate the six most common circovirus subtypes in HSCT patients: TTV-29, TTV-16, TTV-15, TTV-24, TTV-1, and TTV-19, thus significantly improving detection efficiency. Furthermore, the specific primer set and specific probe described in this application are designed specifically for these six circovirus subtypes, targeting only the corresponding specific TTV subtypes, exhibiting high specificity, avoiding cross-amplification, and ensuring the authenticity and interpretability of Ct values ​​for each subtype in the multiplex system. This effectively solves the problems of low detection efficiency and insufficient typing ability in existing technologies. The six circovirus subtypes described in this invention are closely related to the clinical characteristics of HSCT patients; therefore, the detection method described in this invention has greater clinical value than general detection methods, is simple, and has good scalability. Attached Figure Description

[0030] Figure 1 A maximum likelihood phylogenetic tree constructed based on the ViralZone database;

[0031] Figure 2 The dominant circovirus species / subtypes detected in blood and stool samples ( Figure 2 The first line in the table represents the test results for the blood sample, and the second line represents the test results for the stool sample.

[0032] Figure 3 A time-varying heatmap of the circovirus subtypes detected in patient C. Figure 3 The background colors represent sample types or specific events: white represents plasma, light gray represents feces, green represents donor plasma, and dark gray represents time points where the environment changed or data was missing; dark and larger circles indicate that the relative abundance of the corresponding circular virus subtype was higher at each time point. Figure 3 In this context, C1-C40 represent different time periods within a year. Detailed Implementation

[0033] This invention provides a specific primer set and specific probe for simultaneously detecting and distinguishing six circovirus subtypes.

[0034] In this invention, the six circovirus subtypes include TTV-29, TTV-16, TTV-15, TTV-24, TTV-1, and TTV-19. The specific primer sets in this invention include specific primer sets for TTV-29, TTV-16, TTV-15, TTV-24, TTV-1, and TTV-19.

[0035] In this invention, the TTV-29 subtype-specific primer set includes a forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO:1, specifically 5'-ACCTTCTCCTTAGAAGGCATTTAT-3', with a length of 24 bp, a Tm of approximately 61.9 °C, and a GC of approximately 37.5%. The sequence of the reverse primer is shown in SEQ ID NO:2, specifically 5'-TAGGTCTAGGTCATGGTTGGA-3', with a length of 21 bp, a Tm of approximately 61.9 °C, and a GC of approximately 47.6%.

[0036] In this invention, the TTV-16 subtype-specific primer set includes a forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO:4, specifically 5'-CGGCGGAAGCTACACAAA-3', with a length of 18 bp, a Tm of approximately 62.3 °C, and a GC of approximately 55.6%. The sequence of the reverse primer is shown in SEQ ID NO:5, specifically 5'-GGCCTACTTCCGGGTTACA-3', with a length of 19 bp, a Tm of approximately 63.1 °C, and a GC of approximately 57.9%.

[0037] In this invention, the TTV-15 subtype-specific primer set includes a forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO:7, specifically 5'-GGACCGTTTGCATACAGAGA-3', with a length of 20 bp, a Tm of approximately 61.8 °C, and a GC content of approximately 50%. The sequence of the reverse primer is shown in SEQ ID NO:8, specifically 5'-TGACAACCTGGTGGTAGATTAAG-3', with a length of 23 bp, a Tm of approximately 61.9 °C, and a GC content of approximately 43.5%.

[0038] In this invention, the TTV-24 subtype-specific primer set includes a forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO:10, specifically 5'-GGACACCAACTCCGATTCA-3', with a length of 19 bp, a Tm of approximately 61.5 °C, and a GC of approximately 52.6%. The sequence of the reverse primer is shown in SEQ ID NO:11, specifically 5'-GGTCGCGGGCCTAATAAT-3', with a length of 18 bp, a Tm of approximately 61.5 °C, and a GC of approximately 55.6%.

[0039] In this invention, the TTV-1 subtype-specific primer set includes a forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO:13, specifically 5'-GGAAGCGTCTCCACTATACAC-3', with a length of 21 bp, a Tm of approximately 61.7 °C, and a GC of approximately 52.4%. The sequence of the reverse primer is shown in SEQ ID NO:14, specifically 5'-GACGTAGCTGTGACCTTTGA-3', with a length of 20 bp, a Tm of approximately 61.7 °C, and a GC of approximately 50%.

[0040] In this invention, the TTV-19 subtype-specific primer set includes a forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO:16, specifically 5'-GCAGCTCGGCATATACAAA-3', with a length of 19 bp, a Tm of approximately 60.2 °C, and a GC content of approximately 47.4%. The sequence of the reverse primer is shown in SEQ ID NO:17, specifically 5'-GATTGACTTCCGGGCTATAC-3', with a length of 20 bp, a Tm of approximately 59.6 °C, and a GC content of approximately 50%.

[0041] In this invention, the specific probes include TTV29-P, TTV16-P, TTV15-P, TTV24-P, TTV1-P, and TTV19-P. The sequence of TTV29-P is shown in SEQ ID NO:3, specifically 5'-TTTCTGTACCACAGAAACAGGTGG-3', with a length of 24 bp, a Tm of approximately 64.6 °C, and a GC of approximately 45.8%. The 5' end of this sequence is labeled with the fluorescent reporter group Texas Red, and the 3' end is labeled with the NFQ-MGB group. The sequence of TTV16-P is shown in SEQ ID NO:6, specifically 5'-ATGACGTGTGCACGACCTTTGACC-3', with a length of 24 bp, a Tm of approximately 68.7 °C, and a GC of approximately 54.2%. The 5' end of this sequence is labeled with the fluorescent reporter group VIC, and the 3' end is labeled with the NFQ-MGB group. The sequence of TTV15-P described in this invention is shown in SEQ ID NO:9, specifically 5'-AGACTGCAAGCTGACTTGACTGCA-3', with a length of 24 bp, a Tm of approximately 68.2 °C, and a GC of 50%. The 5' end of this sequence is labeled with the fluorescent reporter group FAM, and the 3' end is labeled with the NFQ-MGB group. The sequence of TTV24-P described in this invention is shown in SEQ ID NO:12, specifically 5'-AAGCGGGTCTCCACCTAAACCC-3', with a length of 22 bp, a Tm of approximately 67.8 °C, and a GC of approximately 59.1%. The 5' end of this sequence is labeled with the fluorescent reporter group Texas Red, and the 3' end is labeled with the NFQ-MGB group. The sequence of TTV1-P described in this invention is shown in SEQ ID NO:15, specifically 5'-TTGCCCAAGATGGCGGCAAA-3', with a length of 20 bp, a Tm of approximately 67.7 °C, and a GC of 55%. The 5' end of this sequence is labeled with the fluorescent reporter group VIC, and the 3' end is labeled with the NFQ-MGB group. The sequence of TTV19-P described in this invention is shown in SEQ ID NO:18, specifically 5'-TGACGTAGGTGTGACCTATGACCC-3', with a length of 24 bp, a Tm of approximately 66.7 °C, and a GC of approximately 54.2%. The 5' end of this sequence is labeled with the fluorescent reporter group FAM, and the 3' end is labeled with the NFQ-MGB group.

[0042] This invention also provides the application of the specific primer set and specific probe in the preparation of products that simultaneously detect and distinguish six circovirus subtypes.

[0043] In this invention, the product includes a reagent kit.

[0044] The present invention also provides a kit for simultaneously detecting and distinguishing six circovirus subtypes, including the aforementioned specific primer set and specific probe.

[0045] The present invention also provides a method for simultaneously detecting and differentiating six circovirus subtypes for purposes other than disease diagnosis and / or treatment, comprising the following steps:

[0046] (1) Extract DNA from the sample to be tested to obtain a DNA sample;

[0047] (2) The DNA sample from step (1) is amplified by qPC using the specific primer set and specific probe, and the results are determined based on the fluorescence curve and Ct value of the amplification product.

[0048] In this invention, the qPC amplification system further includes an internal reference primer set and an internal reference probe. The internal reference primer set consists of a forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO.19, specifically 5'-GTCACTCCACTCCCATGTC-3'; the sequence of the reverse primer is shown in SEQ ID NO.20, specifically 5'-GTTCTCTGGGAACTCACCTC-3'. The internal reference probe of this invention is HPRT1-P, with the sequence shown in SEQ ID NO.21, specifically 5'-TCTGGCCCTAGTCTCAGACCTTCC-3'.

[0049] In this invention, the 5' end of the internal reference probe is connected to a VIC group, and the 3' end is connected to NFQ-MGB.

[0050] In this invention, the amplification system comprises: 10 μL of 2× qPCR premix, 0.3 μL each of 0.3 μM specific forward primers, 0.3 μL each of 0.3 μM specific reverse primers, 0.3 μL of 0.3 μM internal control forward primer, 0.3 μL of 0.3 μM internal control reverse primer, 0.2 μL each of 0.10 μM specific probes, 0.2 μL of 0.10 μM internal control probe, 2 μL of template DNA, and finally, nuclease-free water to a final volume of 20 μL.

[0051] In this invention, the amplification procedure is as follows: pre-denaturation at 95°C for 3 min; 95°C for 15 s, 60°C for 30 s, 40 cycles; 72°C for 30 s.

[0052] In this invention, the signals of each fluorescence channel are read after the amplification is completed. For any target channel, if a typical S-shaped amplification curve appears and Ct≤38, the TTV subtype corresponding to that channel is determined to be positive; if there is no S-shaped amplification curve or Ct>38, it is determined to be negative; if multiple channels are positive simultaneously, it is determined to be a co-detection of multiple subtypes. The negative control should have no amplification signal; the internal control channel should show an amplification signal to verify the effectiveness of the extraction and amplification system.

[0053] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0054] Example 1

[0055] (a) Target subtype and target region selection

[0056] Based on the database (GenBank) and existing plasma / serum virome sequencing results of HSCT patients (HSCT patients were selected from acute leukemia patients aged 18-60 years who visited Peking University People's Hospital and signed informed consent forms), the detection rate and abundance level of different TTV subtypes were statistically analyzed. At least 6 subtypes with the highest detection rate and the closest relationship with clinical outcomes in the HSCT population were selected, namely TTV-29, TTV-24, TTV-16, TTV-1, TTV-15, and TTV-19.

[0057] Full-length genome sequences of the aforementioned subtypes were collected. The genome sequence numbers for the TTV-29 subtype are AB038621, TTV-24 subtype AB060597, TTV-16 subtype NC_014091, TTV-1 subtype MH017570, TTV-15 subtype NC_014096, and TTV-19 subtype NC_014078. Multiple sequence alignment was performed using multiple sequence alignment software (ClustalW, MAFFT).

[0058] Locate the conserved internal promoter TATA box sequence (ATATAA) within each subtype. From the downstream region of the TATA box (within tens to hundreds of bases), identify a fragment with a highly conserved framework but different internal sequences among different subtypes by comparison, and use it as the target region for primer and probe design (subtype-specific amplification target fragment). The target fragments for the TTV-29 subtype are bases 1038 to 1118 of the TTV-29 subtype genome; for the TTV-24 subtype, bases 2677 to 2762 of the TTV-24 subtype genome; for the TTV-16 subtype, bases 3516 to 3618 of the TTV-16 subtype genome; for the TTV-1 subtype, bases 3446 to 3542 of the TTV-1 subtype genome; for the TTV-15 subtype, bases 2292 to 2394 of the TTV-15 subtype genome; and for the TTV-19 subtype, bases 3471 to 3606 of the TTV-19 subtype genome.

[0059] This target region establishment operation, while ensuring that the amplification sites of different TTV subtypes are relatively consistent and have functional homology, utilizes internal variations to achieve unified scanning and subtype differentiation, which is beneficial for multiplex detection and facilitates the establishment of subsequent clinical risk stratification models.

[0060] (II) Primer Design

[0061] Based on the aforementioned target fragments, primers and probes were designed according to the principles of primer length (18-25 bp), primer Tm value (58-65℃), GC content (40-60%, avoiding more than 4 consecutive G or C molecules), probe length (18-30 bp), and probe Tm value (3-10℃ higher than the corresponding primer). The HPRT gene was used as an internal control, and internal control primers and probes were designed accordingly. The 5' end of the internal control probe was labeled with VIC, and the 3' end with NFQ-MGB. Specific primer and probe sequences are shown in Table 1.

[0062] Table 1 Primer and probe sequence information and their numbers

[0063]

[0064]

[0065] Example 2

[0066] Plasma samples were centrifuged from plasma samples collected during the follow-up phase of 15 patients who underwent hematopoietic stem cell transplantation (HSCT) (patients aged 18-60 years with acute leukemia who visited Peking University People's Hospital and signed informed consent forms). Fecal samples were also collected, and nucleic acids were extracted using a viral DNA extraction kit (PureLink™ Viral RNA / DNA Mini Kit, Invitrogen / Life Technologies). The obtained nucleic acids were used for subsequent virological next-generation sequencing analysis.

[0067] Library construction was performed using extracted nucleic acids as templates, and paired-end sequencing was performed on the Illumina sequencing platform with a read length of PE150. The sequencing data volume of each sample was controlled to be approximately 6 GB of raw data to meet the requirements for detecting low-abundance viral signals.

[0068] The sequencing data underwent quality control, and low-quality and adapter sequences were removed. The sequenced sequences were then aligned with the human genome reference sequence, and host-derived sequences were removed. The retained non-host sequences were annotated with viral classification, and further alignment was performed to confirm the results for Anelloviridae (circoviridae) sequences. The obtained TTV-related sequences were compared with the TTV reference sequence database, and subtype assignment was completed based on sequence similarity and phylogenetic / typing rules (e.g.,...). Figure 1 ), to obtain the detection results of TTV subtypes and the relative abundance / dominant subtype information in each sample (e.g. Figure 2 ).

[0069] The results showed that multiple TTV subtypes could be detected in plasma samples from HSCT patients during follow-up, including common subtypes such as TTV-29, TTV-24, TTV-16, TTV-1, TTV-15, and TTV-19; and differences in the dominant subtype were observed among different patients and at different follow-up time points (e.g., Figure 3 The second-generation sequencing genotyping results established in this way serve as a reference for subsequent multiplex qPCR genotyping validation, and are used to evaluate the consistency and reproducibility of the multiplex qPCR genotyping results.

[0070] Example 3: Clinical Sample Validation

[0071] Plasma was collected from plasma samples of 10 other patients who underwent hematopoietic stem cell transplantation (HSCT) (patients aged 18-60 years with acute leukemia who received informed consent at Peking University People's Hospital) during the follow-up phase. Peripheral blood (5 mL) was collected from each patient, and plasma was separated by centrifugation. 200 μL of plasma was collected from each sample, and nucleic acid was extracted using a PureLink™ Viral RNA / DNA Mini Kit (Invitrogen / Life Technologies), with an elution volume of 50 μL. The extracted DNA was used as a template for multiplex qPCR. Multiplex qPCR amplification was performed using the primers and probes designed in Example 1 within the SYBR Green real-time quantitative PCR system. A template-free negative control and known TTV-positive plasma or standards were also included as positive controls.

[0072] The qPCR amplification system includes: 10 μL of 2× qPCR premix, 0.3 μL each of 0.3 μM specific forward primers, 0.3 μL each of 0.3 μM specific reverse primers, 0.3 μL of 0.3 μM internal control forward primer, 0.3 μL of 0.3 μM internal control reverse primer, 0.2 μL each of 0.10 μM specific probes, 0.2 μL of 0.10 μM internal control probe, 2 μL of template DNA, and finally, nuclease-free water to a final volume of 20 μL.

[0073] The amplification procedure was as follows: pre-denaturation at 95℃ for 3 min; 95℃ for 15 s, 60℃ for 30 s, 40 cycles; 72℃ for 30 s.

[0074] In the SYBR Green system, melting curve analysis was performed after amplification. Fluorescence signals were collected every 0.3-0.5℃ from 65℃ to 95℃ to plot melting curves, and the Ct values, curve morphology, and melting peaks of each subtype in the multiplex system were evaluated. In the SYBR Green melting curves, due to differences in amplified fragment length and GC content, TTV-29, TTV-24, TTV-16, TTV-1, TTV-15, and TTV-19 all exhibited distinguishable characteristic melting peaks. Based on the sequencing results of Example 2, when the degree of matching between the test sequence and the reference sequence of a certain subtype is significantly higher than that of other subtypes, and a preset threshold is met, the subtype is determined to exist.

[0075] The results showed that a typical S-shaped amplification curve appeared in any target channel and Ct≤38, indicating that the TTV subtype corresponding to each channel was positive. This means that the present invention has successfully established a multiplex qPCR system that can simultaneously detect and distinguish six microcircular virus subtypes: TTV-29, TTV-24, TTV-16, TTV-1, TTV-15, and TTV-19.

[0076] Comparing the typing results of multiplex qPCR with the Anelloviridae subtype profile obtained from previous virome sequencing, it was found that there was a high degree of consistency in the determination of whether it was positive and the main dominant subtype, with a consistency rate of over 90%.

[0077] When multiple time points were tested on some patients, it was found that among the six subtypes TTV-29, TTV-24, TTV-16, TTV-1, TTV-15 and TTV-19, the dominant subtype could change at different stages. The trend of the dominant subtype change detected at different time points was consistent with the results of virome sequencing and phylogenetic analysis of the patients.

[0078] In summary, the qPCR amplification detection method of this invention, utilizing the specific primer set and specific probe, can simultaneously detect and distinguish six common anisoviral subtypes: TTV-29, TTV-24, TTV-16, TTV-1, TTV-15, and TTV-19. It offers rapid detection and high specificity, effectively solving the problems of low detection efficiency and insufficient typing ability in existing technologies. Furthermore, it can be used for rapid quantification and dynamic monitoring of the Anelloviridae population structure in HSCT patients, providing a feasible tool for assessing patient immune status and clinical risk. Sequencing detection methods are costly and time-consuming, while the qPCR amplification detection method of this invention reduces costs, shortens detection time, and ensures accuracy, making it more acceptable to patients.

[0079] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A specific primer set and specific probe for simultaneously detecting and distinguishing six circovirus subtypes, characterized in that, The circovirus subtypes include TTV-29, TTV-16, TTV-15, TTV-24, TTV-1, and TTV-19. The specific primer sets include TTV-29 subtype specific primer sets, TTV-16 subtype specific primer sets, TTV-15 subtype specific primer sets, TTV-24 subtype specific primer sets, TTV-1 subtype specific primer sets, and TTV-19 subtype specific primer sets. The TTV-29 subtype-specific primer set includes a forward primer with the sequence shown in SEQ ID NO:1 and a reverse primer with the sequence shown in SEQ ID NO:2; The TTV-16 subtype-specific primer set includes a forward primer with a sequence as shown in SEQ ID NO:4 and a reverse primer with a sequence as shown in SEQ ID NO:5; The TTV-15 subtype-specific primer set includes a forward primer with a sequence as shown in SEQ ID NO:7 and a reverse primer with a sequence as shown in SEQ ID NO:8; The TTV-24 subtype-specific primer set includes a forward primer with a sequence as shown in SEQ ID NO:10 and a reverse primer with a sequence as shown in SEQ ID NO:11; The TTV-1 subtype-specific primer set includes a forward primer with a sequence as shown in SEQ ID NO:13 and a reverse primer with a sequence as shown in SEQ ID NO:14; The TTV-19 subtype-specific primer set includes a forward primer with a sequence as shown in SEQ ID NO:16 and a reverse primer with a sequence as shown in SEQ ID NO:17; The specific probes include TTV29-P with the sequence shown in SEQ ID NO:3, TTV16-P with the sequence shown in SEQ ID NO:6, TTV15-P with the sequence shown in SEQ ID NO:9, TTV24-P with the sequence shown in SEQ ID NO:12, TTV1-P with the sequence shown in SEQ ID NO:15, and TTV19-P with the sequence shown in SEQ ID NO:18; The specific probe is labeled with a fluorescent reporter group at its 5' end and an NFQ-MGB group at its 3' end; The fluorescent reporter groups include Texas Red, VIC, FAM, and Cy5; The 5' end of the specific probe TTV29-P sequence is labeled with the fluorescent reporter group Texas Red, the 5' end of the specific probe TTV16-P sequence is labeled with the fluorescent reporter group VIC, the 5' end of the specific probe TTV15-P sequence is labeled with the fluorescent reporter group FAM, the 5' end of the specific probe TTV24-P sequence is labeled with the fluorescent reporter group Texas Red, the 5' end of the specific probe TTV1-P sequence is labeled with the fluorescent reporter group VIC, and the 5' end of the specific probe TTV19-P sequence is labeled with the fluorescent reporter group FAM.

2. The application of the specific primer set and specific probe described in claim 1 in the preparation of a product that can simultaneously detect and distinguish six circovirus subtypes.

3. The application according to claim 2, characterized in that, The product includes a reagent kit.

4. A kit for simultaneously detecting and distinguishing six circovirus subtypes, characterized in that, It includes the specific primer set and specific probe as described in claim 1.

5. A method for simultaneously detecting and differentiating six circovirus subtypes for purposes other than disease diagnosis and / or treatment, characterized in that, Includes the following steps: (1) Extract DNA from the sample to be tested to obtain a DNA sample; (2) The DNA sample described in step (1) is amplified by qPCR using the specific primer set and specific probe described in claim 1, and the results are determined based on the fluorescence curve and Ct value of the amplification product. A positive result is indicated by the appearance of an S-shaped fluorescence curve and a Ct value ≤ 38; a negative result is indicated by a Ct value > 38 or the absence of an S-shaped curve.

6. The method according to claim 5, characterized in that, The qPCR amplification system also includes an internal reference primer set and an internal reference probe. The internal reference primer set consists of a forward primer with the sequence shown in SEQ ID NO. 19 and a reverse primer with the sequence shown in SEQ ID NO.

20. The internal reference probe is HPRT1-P with the sequence shown in SEQ ID NO.

21.

7. The method according to claim 6, characterized in that, The internal reference probe has a VIC group attached to its 5' end and an NFQ-MGB attached to its 3' end.

8. The method according to claim 5, characterized in that, The amplification system includes: 10 μL of 2× qPCR premix, 0.3 μL each of 0.15 μM specific forward primers, 0.3 μL each of 0.15 μM specific reverse primers, 0.3 μL of 0.15 μM internal control forward primer, 0.3 μL of 0.15 μM internal control reverse primer, 0.2 μL each of 0.10 μM specific probes, 0.2 μL of 0.10 μM internal control probe, 2 μL of template DNA, and finally, nuclease-free water to a final volume of 20 μL.

9. The method according to claim 5, characterized in that, The amplification procedure was as follows: pre-denaturation at 95℃ for 3 min; 95℃ for 15 s, 60℃ for 30 s, 40 cycles; 72℃ for 30 s.

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