Primer probe composition and kit for detecting Databan virus and application of primer probe composition and kit for detecting Databan virus

Abstract

The invention provides a primer probe composition and a kit for detecting a Databean virus and application, and relates to the technical field of biological detection, the primer probe composition comprises a primer probe group aiming at a Databean virus S gene and a primer probe group aiming at a Databean virus L gene, the Databean virus L and S genes are used as amplification target genes for design, and the kit is used for detecting the Databean virus. The primer probe group aiming at the S gene of the Databu virus and the primer probe group aiming at the L gene of the Databu virus are used as the primer probe composition, so that multi-gene target amplification is realized, and the positive detection rate reaches 100% and is higher than the positive detection rate of single-gene amplification; the lowest detection limit is up to 200 copies / mL; the quantification limit reaches 500 copies / mL, and the quantification linear range is 5.0 * 10 < 2 >-1.0 * 10 < 8 > copies / mL. The technical problem that in the prior art, a high-sensitivity and high-accuracy multiplex qPCR detection method for the nucleic acid of the copyda virus is lacked is solved.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a primer-probe combination and a kit and application thereof for detecting Dabiebandha virus. Background Art

[0002] Dabie bandavirus (DBV), formerly known as severe fever with thrombocytopenia syndrome virus (SFTSV), belongs to the genus Phlebovirus in the family Bunyaviridae. The DBV genome consists of three single-stranded negative-sense RNA fragments: large (L), medium (M), and small (S). DBV is divided into five sublineages (AE). DBV directly acts on a variety of human cells to cause tissue and organ damage. The virus attacks the human lymph nodes, causing lymphadenopathy and necrotizing lymphadenitis. After rapid replication in the lymph nodes and spleen, it enters the systemic circulation, forming viremia and attacking multiple tissues and organs at the same time. DBV infection leads to immune dysfunction. In severe cases, it can induce cytokine storms and endothelial damage. Patients may die from bleeding or multiple organ failure. At present, DBV infection cases have been reported in 25 provinces and cities in China. It is mainly prevalent in Asia. Infection has also been reported in central and eastern China, South Korea, and Japan in East Asia. In recent years, the number of DBV infection cases has steadily increased. Dabiebanda virus is mainly transmitted through the bites of infected vectors such as the long-horned tick. It can also cause infection through unprotected contact with the blood, secretions, excrement and contaminants of infected animals or patients.

[0003] At present, clinical diagnosis of Dabiebanda virus infection is mainly through three ways: nucleic acid detection, virus culture and serological detection. Virus isolation and culture is the "gold standard" for virus detection, but the operation process is frequent, the isolation and culture of the virus takes a long time, generally requiring 1 to 2 weeks, and the detection efficiency is low, which is not suitable for the early differential diagnosis of the virus. In addition, there are no strict standardized operating standards for virus culture, and the results of virus isolation vary when operated by different personnel. Although serological testing is simple to operate, in the early stages of viral infection, the antibody titer produced in the patient's body is generally low and false negatives are prone to occur, which is not conducive to the early differential diagnosis of viral infection.

[0004] Viral nucleic acid detection is the most direct, sensitive and specific detection indicator for viral infection. The real-time fluorescence qRT-PCR method is widely used in the detection of microbial nucleic acids because of its sensitivity, specificity, and quantitative accuracy. Multiplex qPCR requires the use of probe-based assays. By adding more than two pairs of specific primers to the same system, multiple target fragments can be amplified from the same system to prevent false negatives caused by gene mutations or partial nucleic acid degradation, thereby improving the accuracy and reliability of the detection. In addition, multiplex qPCR has higher sensitivity for simultaneous amplification of multiple sites than single-plex qPCR. However, due to the mutual influence of multiple target genes in the design of multiplex qPCR, improper design of primer and probe sequences may lead to non-specific amplification. Therefore, the primer and probe need to be optimized to achieve the best amplification effect.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the purposes of the present invention is to provide a primer-probe combination for detecting Dabiebandha virus, so as to solve the technical problem of the lack of a multiplex qPCR detection method for Dabiebandha virus nucleic acid with high sensitivity and accuracy in the prior art.

[0007] The second object of the present invention is to provide the use of the primer-probe combination mentioned above in the preparation of a product for detecting Bandar Virus.

[0008] The third object of the present invention is to provide a reagent for detecting the Dabiebandha virus.

[0009] A fourth object of the present invention is to provide a kit for detecting Bandovirus.

[0010] A fifth object of the present invention is to provide the use of the above reagent or the above kit in detecting Bandhavn virus for non-diagnostic purposes.

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0012] In a first aspect, the present invention provides a primer-probe combination for detecting Dabiebanda virus, comprising a primer-probe set for the Dabiebanda virus S gene and a primer-probe set for the Dabiebanda virus L gene;

[0013] The primer probe set for the S gene of Dabiebandha virus comprises a forward primer, a reverse primer and a probe, and the nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively;

[0014] The primer probe set for the L gene of the Dabiebandha virus includes a forward primer, a reverse primer and a probe, and the nucleotide sequences are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0015] Furthermore, it also includes an internal reference gene primer probe set, which includes a forward primer, a reverse primer and a probe, and the nucleotide sequences are shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 respectively.

[0016] Furthermore, the probe for the Dabiebandha virus S gene, the probe for the Dabiebandha virus L gene and the probe for the internal reference gene are respectively labeled with different fluorescent reporter groups.

[0017] Furthermore, the 5' end of the probe contains a fluorescent reporter group, and the 3' end of the probe contains a fluorescent quencher group;

[0018] The fluorescent reporter group includes FAM, VIC, TET, JOE, HEX, CY3, CY5.5, TAMRA, ROX, TexasRed, CY5 or CY7;

[0019] The fluorescence quenching group includes BHQ1, BHQ2, BHQ3 or MGB.

[0020] In a second aspect, the present invention provides the use of the primer-probe combination described above in the preparation of a product for detecting Bandovirus.

[0021] In a third aspect, the present invention provides a reagent for detecting the Dabiebandha virus, comprising the above-mentioned primer-probe combination.

[0022] In a fourth aspect, the present invention provides a kit for detecting Bandovirus, comprising the above-mentioned primer-probe combination or the above-mentioned reagent.

[0023] Furthermore, the invention also includes a PCR reaction solution, wherein the PCR reaction solution includes DNA polymerase, reverse transcriptase, RNase inhibitor, Mg 2+ , PCR buffer and dNTPs.

[0024] In a fifth aspect, the present invention provides the use of the above reagent or the above kit in detecting Bandhavnidae virus for non-diagnostic purposes.

[0025] Further, the application includes applying a primer probe set for the Dabiebandha virus S gene and a primer probe set for the Dabiebandha virus L gene to an amplification system;

[0026] Preferably, the method further comprises applying an internal reference gene primer probe set to the amplification system as an internal reference.

[0027] The present invention provides a primer-probe combination for detecting Dabiebanda virus. The Dabiebanda virus L and S genes are designed as amplification target genes. A primer-probe group targeting the Dabiebanda virus S gene and a primer-probe group targeting the Dabiebanda virus L gene are used as primer-probe combinations, thereby realizing multi-gene target amplification. The positive detection rate reaches 100%, which is higher than the positive detection rate of single gene amplification. The minimum detection limit reaches 200 copies / mL; the quantitative limit reaches 500 copies / mL, and the quantitative linear range is 5.0×10 2 ~1.0×10 8 The invention solves the technical problem that the prior art lacks a multiplex qPCR detection method for Dabiebandha virus nucleic acid with high sensitivity and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 The quantitative linear range of a kit for detecting Dabiebanda virus provided in Example 7 of the present invention;

[0030] Figure 2 The comparison results of clinical sample testing of different kits provided in Example 8 of the present invention. DETAILED DESCRIPTION

[0031] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0032] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0033] In one aspect, the present invention provides a primer-probe combination for detecting Dabiebanda virus, comprising a primer-probe group targeting the Dabiebanda virus S gene and a primer-probe group targeting the Dabiebanda virus L gene;

[0034] The primer probe set for the S gene of Dabiebandha virus comprises a forward primer, a reverse primer and a probe, and the nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively;

[0035] The primer probe set for the L gene of the Dabiebandha virus includes a forward primer, a reverse primer and a probe, and the nucleotide sequences are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0036] The primer-probe combination for detecting Dabiebanda virus of the present invention is designed with Dabiebanda virus L and S genes as amplification target genes, and a primer-probe group for Dabiebanda virus S gene and a primer-probe group for Dabiebanda virus L gene are used as primer-probe combinations, so that multi-gene target amplification is achieved, and the positive detection rate reaches 100%, which is higher than the positive detection rate of single gene amplification; the minimum detection limit reaches 200 copies / mL; the quantitative limit reaches 500 copies / mL, and the quantitative linear range is 5.0×10 2 ~1.0×10 8 The invention solves the technical problem that the prior art lacks a multiplex qPCR detection method for Dabiebandha virus nucleic acid with high sensitivity and accuracy.

[0037] In some specific embodiments, an internal reference gene primer probe set is also included, and the internal reference gene primer probe set includes a forward primer, a reverse primer and a probe, and the nucleotide sequences are shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 respectively.

[0038] An internal reference gene primer probe set was designed for the internal reference gene, which is an exogenous gene and can avoid false negative results and prevent the exogenous internal reference from affecting the results.

[0039] In some specific embodiments, the probe targeting the Dabiebandha virus S gene, the probe targeting the Dabiebandha virus L gene, and the probe of the internal reference gene are respectively labeled with different fluorescent reporter groups.

[0040] The two probes in the primer probe set for the Dabiebanda virus S gene and the primer probe set for the Dabiebanda virus L gene are set to different fluorescent reporter groups to achieve accurate detection of the Dabiebanda virus L gene and the Dabiebanda virus S gene, thereby improving the sensitivity and accuracy of detecting the Dabiebanda virus.

[0041] In some specific embodiments, the 5' end of the probe contains a fluorescent reporter group, and the 3' end of the probe contains a fluorescent quencher group; the fluorescent reporter group includes but is not limited to FAM, VIC, TET, JOE, HEX, CY3, CY5.5, TAMRA, ROX, Texas Red, CY5 or CY7; the fluorescent quencher group includes but is not limited to BHQ1, BHQ2, BHQ3 or MGB. The fluorescent reporter group and the fluorescent quencher group are labeled using conventional labeling methods in the art.

[0042] According to another aspect of the present invention, there is also provided use of the primer-probe combination described above in the preparation of a product for detecting the Dabiebanda virus.

[0043] According to another aspect of the present invention, a reagent for detecting the Bepanda virus is provided, comprising the above-mentioned primer-probe combination.

[0044] According to another aspect of the present invention, a kit for detecting the Dabiebanda virus is provided, comprising the above-mentioned primer-probe combination or the above-mentioned reagent.

[0045] Experiments have shown that the minimum detection limit of the kit is 200 copies / mL, the quantitative limit is 500 copies / mL, and the quantitative linear range is 5.0×10 2 ~1.0×10 8 copies / mL, the positive detection rate was 100%; and it had good repeatability.

[0046] In some specific embodiments, a PCR reaction solution is also included, which includes but is not limited to DNA polymerase, reverse transcriptase, RNase inhibitor, Mg 2+ , PCR buffer and dNTPs. When detecting Dabiebanda virus, the concentration range of primers and probes in the amplification system of the kit of the present invention is shown in Table 1.

[0047] Table 1 Optional range of primer and probe concentrations in the amplification system

[0048] Reagents Concentration (μM) Upstream primer F1 0.02～20 Downstream primer R1 0.02～20 Probe P1 0.01～10 Upstream primer F2 0.02～20 Downstream primer R2 0.02～20 Probe P2 0.01～10 Upstream primer F3 0.02～20 Downstream primer R3 0.02～20 Probe P3 0.01～10

[0049] According to another aspect of the present invention, there is also provided the use of the above reagent or the above kit in detecting Bandhavnidae virus for non-diagnostic purposes.

[0050] In some specific embodiments, a primer probe set for the Dabie Bandha virus S gene and a primer probe set for the Dabie Bandha virus L gene are applied to the amplification system; through multiple detections of the Dabie Bandha virus S gene and the Dabie Bandha virus L gene, efficient and accurate Dabie Bandha virus detection is achieved, which can be applied to the detection of a large number of samples and is convenient for clinical application.

[0051] In some specific embodiments, an internal reference gene primer probe set is also applied to the amplification system as an internal reference.

[0052] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] Example 1 Dabiebandha virus specific primer probe composition

[0054] In this embodiment, the disclosed L gene and S gene of Dabiebandha virus are used as amplification target genes, and specific primers and fluorescent probes are analyzed and designed. The design of primers and probes is assisted by design software such as Primer Premier5.0 and Oligo 6.0. The designed primer probes are all compared by homology by BLAST to ensure their specificity. Primers and probes are synthesized by Shanghai Shenggong Bioengineering Co., Ltd. The primer-probe combination includes 2 pairs of primers and 2 probes for quantitative detection of Dabiebandha virus, and also includes 1 pair of primers and 1 probe as an internal control gene primer-probe combination. One-step RT-PCR amplification is carried out, and quantitative detection of Dabiebandha virus RNA in plasma or serum samples is realized by changes in fluorescent signals. The primer-probe group sequence for quantitative detection of Dabiebandha virus is shown in Table 2.

[0055] Table 2 Sequences of the first set of primer-probe combinations

[0056]

[0057] Among them, probe P1 is labeled with Cy5 fluorescent reporter group; P2 is labeled with TR fluorescent reporter group; and probe P3 is labeled with FAM fluorescent reporter group.

[0058] Example 2

[0059] A kit for detecting Dabiebanda virus comprises the primer-probe combination provided in Example 1 and a PCR reaction solution.

[0060] The PCR reaction solution includes DNA polymerase, reverse transcriptase, RNase inhibitor, Mg 2+ , PCR buffer and dNTPs.

[0061] Embodiment 3 Dabiebanda virus detection method

[0062] The sample nucleic acid was used as a template and the kit provided in Example 2 was used to detect the Dabiebandha virus. In this example, the magnetic bead method was used to extract the sample nucleic acid.

[0063] In this example, a 50 μl PCR amplification system was used, the primer and probe concentrations were specifically shown in Table 3, and the amplification conditions were shown in Table 4.

[0064] Table 3 Primer probe concentration settings in the amplification system

[0065] Reagents Concentration (μM) Upstream primer F1 0.2 Downstream primer R1 0.2 Probe P1 0.1 Upstream primer F2 0.2 Downstream primer R2 0.2 Probe P2 0.1 Upstream primer F3 0.2 Downstream primer R3 0.2 Probe P3 0.1

[0066] Table 4 Amplification conditions

[0067]

[0068] Two channels (Cy5 and TR) were used to collect fluorescence in PCR amplification, and a quantitative standard curve was determined for each batch of Dabiebandha virus test kit. The Dabiebandha virus load in the sample was calculated based on the Ct value.

[0069] Example 4: Validation of the minimum detection limit of the kit

[0070] The kit provided in Example 2 was selected and the detection method provided in Example 3 was used. The minimum detection limit verification process was as follows: the minimum detection limit concentration was prepared under negative plasma matrix, 200 copies / mL of 4 common different subtypes (A, B, E, F) of Dabiebandha virus culture were prepared, 10 parallels were made for each, and used for full process detection, and negative plasma without Dabiebandha virus template was used as negative control.

[0071] The minimum detection limit verification experiment showed that when the concentration of Dabiebanda virus was 200 copies / mL, all four types of virus cultures were detected, and the positive rate was 100%. Therefore, the minimum detection limit of Dabiebanda virus in this method was 200 copies / mL. The results are shown in Table 5.

[0072] Table 5 Summary of sample minimum detection limit results

[0073]

[0074]

[0075] Example 5: Test kit quantitative limit verification

[0076] The kit provided in Example 2 was selected and operated according to the detection method provided in Example 3. The quantitative limit verification process of the kit was as follows: the quantitative limit concentration was prepared under the negative plasma matrix, 500 copies / mL of 4 types of Dabiebanda virus culture were prepared, and 5 parallels were made for each, which were used for full-process detection, and negative plasma without Dabiebanda virus template was used as a negative control.

[0077] The quantitative limit verification experiment showed that when the concentration of Dabiebanda virus was 500 copies / mL, the positive rate of the four types of Dabiebanda virus cultures was 100%, and the logarithmic difference between the test concentration and the actual concentration was ≤0.5. Therefore, the quantitative limit of Dabiebanda virus in this method is 500 copies / mL. The results are shown in Table 6.

[0078] Table 6 Summary of sample quantitation limits

[0079]

[0080]

[0081] Example 6 Kit Repeatability

[0082] The kit provided in Example 2 was selected and the detection method provided in Example 3 was used. Four types of Bandovirus were diluted to 5000 copies / mL with negative plasma matrix, and the test was repeated 10 times. The CV of the logarithmic value of the test concentration was calculated. The results showed that the CV of the logarithmic value of the concentration was ≤5%, and the results are shown in Tables 7 and 8.

[0083] Table 7 Intra-batch and inter-batch precision CV of logarithmic values ​​of sample concentration (I)

[0084]

[0085]

[0086] Table 8 Intra-batch and inter-batch precision CV of logarithmic values ​​of sample concentration (II)

[0087]

[0088] Example 7 Kit Quantitative Linear Range

[0089] The kit provided in Example 2 was selected and the detection method provided in Example 3 was used for operation. The Dabiebandha virus type B was diluted with negative plasma matrix to 5.00E+02, 1.00E+03, 2.00E+04, 2.00E+06 and 2.00E+08 copies / mL, and each concentration was repeated 3 times. The logarithm of the test concentration was calculated and the quantitative linear range of the kit was determined. The standard curve was y=1.0678x-0.4666, R 2 =0.9965, such as Figure 1 The results showed that the quantitative linear range of this kit was 5.0×10 2 ~1.0×10 8 copies / mL. The results are shown in Table 9.

[0090] Table 9 Quantitative linear range of the kit

[0091]

[0092]

[0093] Example 8 Detection of clinical samples by the kit

[0094] The kit provided in Example 2 was selected and the detection method provided in Example 3 was used for the operation. The samples were 30 plasma samples and 20 negative plasma samples from patients clinically diagnosed with fever with thrombocytopenia syndrome, collected from Wuhan City, Hubei Province. These 50 clinical samples were tested and compared with the results of the control reagent test. The control reagent was the fever with thrombocytopenia syndrome Bunyavirus nucleic acid detection kit (PCR-fluorescent probe method) of Guangzhou Daan Gene Co., Ltd., with national device registration number 20153402023. The results are as follows Figure 2 As shown, the standard curve is y = 0.985x + 0.0736, R 2 =0.9369, the positive and negative coincidence rates of this kit and the comparative kit were both 100%, and the logarithmic differences of the quantitative concentrations of the two kits were ≤0.5, indicating that the performance consistency of this kit and the comparative kit was good (as shown in Tables 10 and 11).

[0095] Table 10 Analysis of consistency of clinical samples tested with comparative reagents (I)

[0096]

[0097]

[0098] Table 11 Analysis of consistency of clinical samples tested with comparative reagents (II)

[0099]

[0100]

[0101] Example 9 Comparison of different single gene and multi-gene amplification performance

[0102] This embodiment implements three groups of S gene single amplification system, L gene single amplification system and S+L double amplification system respectively. The three groups of amplification systems test low concentration (200 copies / ml) Dabiebanda virus samples respectively. The detection results of different amplification systems are shown in Table 12: the positive detection rates of the S gene single amplification system and the L gene single amplification system are 90% and 80% respectively; the positive detection rate of the S+L double amplification system is 100%, and the results show that the performance of multi-gene amplification is better than that of single gene detection.

[0103] Table 12 Single gene and multi-gene amplification

[0104]

[0105]

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer-probe combination for detecting Dabiebandha virus, characterized in that: It includes a primer probe set for the S gene of Dabiebandha virus and a primer probe set for the L gene of Dabiebandha virus; The primer probe set for the S gene of Dabiebandha virus comprises a forward primer, a reverse primer and a probe, and the nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; The primer probe set for the L gene of the Dabiebandha virus includes a forward primer, a reverse primer and a probe, and the nucleotide sequences are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

2. The primer-probe combination according to claim 1, characterized in that It also includes an internal reference gene primer probe set, which includes a forward primer, a reverse primer and a probe, and the nucleotide sequences are shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 respectively.

3. The primer-probe combination according to claim 2, characterized in that: The probe targeting the Dabiebandha virus S gene, the probe targeting the Dabiebandha virus L gene and the probe of the internal reference gene are respectively labeled with different fluorescent reporter groups.

4. The primer-probe combination according to claim 1, characterized in that: The 5' end of the probe contains a fluorescent reporter group, and the 3' end of the probe contains a fluorescent quencher group; The fluorescent reporter group includes FAM, VIC, TET, JOE, HEX, CY3, CY5, TAMRA, ROX, Texas Red, CY5.5 or CY7; The fluorescence quenching group includes BHQ1, BHQ2, BHQ3 or MGB.

5. Use of the primer-probe combination according to any one of claims 1 to 4 in the preparation of a product for detecting Bepanda virus.

6. A reagent for detecting Dabiebandha virus, characterized in that: The invention comprises the primer-probe combination according to any one of claims 1 to 4.

7. A kit for detecting Dabiebandha virus, characterized in that: The method comprises the primer-probe combination according to any one of claims 1 to 4 or the reagent according to claim 6.

8. The kit according to claim 7, characterized in that The PCR reaction solution also includes DNA polymerase, reverse transcriptase, RNase inhibitor, Mg 2+ , PCR buffer and dNTPs.

9. Use of the reagent according to claim 6 or the kit according to any one of claims 7 or 8 in detecting Bandha virus for non-diagnostic purposes.

10. The use according to claim 9, characterized in that: The application includes applying a primer probe set targeting the S gene of Dabiebandha virus and a primer probe set targeting the L gene of Dabiebandha virus to an amplification system; Preferably, the method further comprises applying an internal reference gene primer probe set to the amplification system as an internal reference.

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