CN120536642A - A kit for on-site detection of monkeypox virus typing and its application and product - Google Patents
A kit for on-site detection of monkeypox virus typing and its application and productInfo
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- CN120536642A CN120536642A CN202511062843.9A CN202511062843A CN120536642A CN 120536642 A CN120536642 A CN 120536642A CN 202511062843 A CN202511062843 A CN 202511062843A CN 120536642 A CN120536642 A CN 120536642A
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Abstract
本发明提供一种现场化检测猴痘病毒分型试剂盒及其应用和产品，属于分子检测技术领域。本发明首先提供了现场化猴痘病毒分型试剂盒，包括用于鉴定猴痘分支Ⅰa型、分支Ⅰb型和分支Ⅱ型的引物探针组合物。本发明进一步还提供了包括猴痘鉴定引物探针或正痘病毒属鉴定引物探针的联合型试剂盒。本发明通过对引物探针的组合优化，达到了同一扩增体系内平衡扩增的效果，一次性对猴痘病毒进行准确分型，并同时实现猴痘病毒或正痘病毒属的鉴定。且本发明的试剂盒具有高灵敏度和高准确性的特点，同时能够减少外来物质的干扰，稳定性强，具有较高的实际应用价值。
The present invention provides a kit for on-site detection of monkeypox virus typing, its application and product, and belongs to the field of molecular detection technology. The present invention first provides an on-site monkeypox virus typing kit, comprising a primer-probe combination for identifying monkeypox clade Ia, clade Ib and clade II. The present invention further provides a combined kit comprising a monkeypox identification primer probe or an orthopoxvirus identification primer probe. The present invention achieves the effect of balanced amplification within the same amplification system by optimizing the combination of primers and probes, accurately typing the monkeypox virus at one time, and simultaneously identifying the monkeypox virus or the orthopoxvirus genus. The kit of the present invention has the characteristics of high sensitivity and high accuracy, while being able to reduce the interference of foreign substances, having strong stability, and having high practical application value.
Description
Technical Field
The invention belongs to the technical field of molecular detection, and particularly relates to a field monkey pox virus typing kit, and application and a product thereof.
Background
Monkey poxvirus (Monkeypox virus, MPXV) belongs to the family poxviridae, genus orthopoxvirus, with a typical morphology of genus orthopoxvirus. The monkey poxvirus is a DNA virus of relatively complex structure. The genome of the monkey poxvirus is double-stranded DNA, approximately 197kb long, and the virus comprises 190 open reading frames. Monkey poxviruses and smallpox viruses, vaccine viruses and vaccinia viruses are 4 viruses of the genus orthopoxvirus that are pathogenic to humans, and all contain soluble antigens, nucleoprotein antigens and hemagglutinin, are essentially identical in antigen form, have cross-immunity to each other, and need to be distinguished for diagnosis.
In addition, monkey poxviruses include different subtypes, and the prior art has disclosed that monkey poxviruses are classified into a branch I type (conventionally called Congo type or medium-non type) and a branch II type (conventionally called Western type), and the same points of the monkey poxbranch I type and the branch II type viruses are mainly that transmission routes are mainly direct contact with secretions and exudates of lesion sites of patients, and symptoms such as rash, fever, lymphadenopathy and the like can be caused after infection. In terms of disease severity, cases of branched type I virus infection have a broader range of rashes, a greater number, and a potentially higher risk of severe and death. The monkey poxvirus comprises a branch type Ia and a branch type Ib under the branch type I, wherein symptoms caused after infection of the branch type Ib variant strain are possibly more serious, and the risks of severe symptoms and death are also higher. Therefore, distinguishing the monkey poxvirus types in the actual detection is beneficial to the adaptive adjustment of the clinical treatment mode.
Confirmation of MPXV infection is based on nucleic acid amplification detection, either by common PCR combined sequencing or by real-time fluorescent quantitative PCR (qPCR). Sequencing has a more accurate result, but has a long sequencing cycle and high cost, and is difficult to realize large-scale application. Therefore, qPCR method is more suitable for clinical large-scale detection.
The detection and typing of the monkey pox virus nucleic acid in one tube are a main research and development direction in the field, but the design area of the primer probe for detecting the monkey pox virus nucleic acid in the prior art lacks good selectivity for other viruses of orthopoxvirus genus, has a certain false positive risk, and the scheme of using a plurality of sets of primer probes increases the cost of reagents, so that the result interpretation is more complex. The multichannel fluorescence PCR is used for rapidly diagnosing and parting the monkey pox virus, and has important significance for epidemic prevention and control and clinical diagnosis and drug administration. In addition, the detection method disclosed in the prior art is mainly used for distinguishing the monkey pox branch I type virus and the monkey pox branch II type virus, and the variant strains Ia and Ib cannot be accurately distinguished, so that a large research and development space is still provided.
The method can detect the monkey pox virus with high specificity, has higher sensitivity, realizes the visualization of the result, avoids the false positive result caused by aerosol, has stable and reliable detection result, and is hopeful to realize the rapid visual detection of epidemic disease sites. However, the parting range is smaller, and there is room for further optimization.
Disclosure of Invention
In order to solve the problems, the invention provides a field monkey pox virus typing kit, which comprises the following primer probe compositions:
the primer is used for typing the monkey poxvirus Ia, and comprises a forward primer mpxIa-F, a reverse primer mpxIa-R, a fluorescent labeled probe mpxIa-P and a fluorescent labeled probe, wherein the forward primer mpxIa-F has a nucleotide sequence shown as SEQ ID NO.1, the reverse primer mpxIa-R has a nucleotide sequence shown as SEQ ID NO.2, and the fluorescent labeled probe mpxIa-P has a nucleotide sequence shown as SEQ ID NO. 3;
The primer is used for typing the monkey pox virus Ib, and comprises a forward primer mpxIb-F, a reverse primer mpxIb-R, a fluorescent labeled probe mpxIb-P and a fluorescent labeled probe mpxIb-P, wherein the forward primer mpxIb-F has a nucleotide sequence shown as SEQ ID NO.4, the reverse primer mpxIb-R has a nucleotide sequence shown as SEQ ID NO.5, and the fluorescent labeled probe mpxIb-P has a nucleotide sequence shown as SEQ ID NO. 6;
The primer is used for typing the monkey pox virus II, and comprises a forward primer mpxII-F, a reverse primer mpxII-R and a fluorescent labeled probe mpxII-P, wherein the forward primer mpxII-F has a nucleotide sequence shown as SEQ ID NO.7, the reverse primer mpxII-R has a nucleotide sequence shown as SEQ ID NO.8, and the fluorescent labeled probe mpxII-P has a nucleotide sequence shown as SEQ ID NO. 9.
Monkey poxvirus Ia typing primer:
mpxIa-F:ATTATCGATATCTCGAGGCGA(SEQ ID NO.1);
mpxIa-R:AAGAATAGTGTAGAGACTGATGCTAA(SEQ ID NO.2);
mpxIa-P:CATTGATTAAAGAGTGTCCATCCGGTAC(SEQ ID NO.3);
monkey poxvirus Ib typing primer:
mpxIb-F:AGAATTTTCTATTTCAACGGGTATAGC(SEQ ID NO.4);
mpxIb-R:TGGATATGCGCCTGAATATCTAG(SEQ ID NO.5);
mpxIb-P:TGAACACGGCACTTCGAAATGGAAA(SEQ ID NO.6);
monkey poxvirus II typing primer:
mpxII-F:GTGGTCCCGGAACATATTCT(SEQ ID NO.7);
mpxII-R:GATTCGCTGAGACCGGTAGTG(SEQ ID NO.8);
mpxII-P:TCAACGACACATCGTGTACTCGGACGA(SEQ ID NO.9);
Optionally, the on-site monkey pox virus typing kit further comprises a PCR amplification system, wherein the PCR amplification system comprises any one or more of dNTPs, DNA polymerase and PCR reaction buffer solution.
Preferably, the PCR amplification system may be a commercial amplification system including, but not limited to, air-Dryable 1-Step RT-qPCR Mix.
Optionally, the on-site monkey pox virus typing kit further comprises reagents for an integrated microfluidic chip, including any one or more of purification reagents, air-drying qPCR reaction reagents and freeze-drying IPC process quality control. The air-drying qPCR reaction reagent comprises a primer probe composition and a PCR amplification system.
Specifically, the purification reagent comprises a lysate, an eluent and magnetic beads, wherein the lysate comprises guanidine hydrochloride, sodium acetate, tritonX-100 and the like, the eluent comprises Tris-HCl and the like, and the magnetic beads can be dried magnetic beads.
The freeze-drying IPC process quality control product comprises a positive quality control product and a freeze-drying system, wherein the positive quality control product can be plasmid, pseudovirus or the like, and the freeze-drying system can comprise mannitol, trehalose, bovine serum albumin, an antifoaming agent, 2-hydroxypropyl-beta-cyclodextrin, tris-HCl, naCl, tween and the like. The freeze-drying IPC process quality control product can be packaged in an integrated micro-fluidic chip in a freeze-drying ball mode. The positive quality control can be a mixture of positive plasmids or pseudoviruses of three subtypes of the monkey pox virus.
In some specific examples, the lysate may be referred to in the art, including 6M guanidine hydrochloride, 0.4M sodium acetate (pH 4.7) and 2% Triton X-100, and the eluate may include 10mM Tris-HCl (pH 8.5).
The specific application of the kit can be realized by a disclosed or unpublished microfluidic chip or an integrated continuous reaction device, such as a continuous reaction device based on nucleic acid extraction, purification and amplification disclosed in the patent with the application number of CN202110057514.0, a microfluidic chip for detecting whole blood nucleic acid disclosed in the patent with the application number of CN201810684320.1, a nucleic acid detection chip for portable equipment disclosed in the patent with the application number of CN201810684021.8, a handheld real-time fluorescence quantitative PCR instrument disclosed in the patent with the application number of CN201930535260.2, a chip device for detecting nucleic acid disclosed in the patent with the application number of CN202110055537.8, or other similar commercial products.
Specifically, the detection method of the kit may include the following steps:
(1) Extracting and purifying sample nucleic acid;
(2) Amplifying the purified nucleic acid of step (1) using the primer probe composition of the present invention;
(3) And analyzing the amplification result.
In some specific embodiments, the above steps may be performed on a microfluidic integrated chip using CarryOn P F rapid nucleic acid detection device, as an example.
Optionally, the monkey poxvirus typing kit further comprises a positive quality control and a negative quality control.
Specifically, the positive quality control product comprises a detection target gene.
On the basis of the monkey pox virus typing kit, the invention also provides a spot monkey pox identification and typing kit.
The on-site monkey pox identification and typing kit comprises all reagents in the monkey pox virus typing kit, and further comprises a primer probe for monkey pox virus identification, a forward primer mpx-F, a reverse primer mpx-R and a fluorescence labeling probe mpx-P, wherein the forward primer mpx-F has a nucleotide sequence shown as SEQ ID NO.10, the reverse primer mpx-R has a nucleotide sequence shown as SEQ ID NO.11, and the fluorescence labeling probe mpx-P has a nucleotide sequence shown as SEQ ID NO. 12.
mpx-F:TGGAGAAGCGAGAAGTTAATAAAGC(SEQ ID NO.10);
mpx-R:CATCATCTATTATAGCATCAGCATC(SEQ ID NO.11);
mpx-P:CCGTGTATCAGCATCCATTGTCGTAGACC(SEQ ID NO.12);
On the basis of the on-site monkey pox virus typing kit, the invention also provides an on-site monkey pox typing combined orthopoxvirus identification kit.
The on-site monkey pox typing combined orthopoxvirus genus identification kit comprises all reagents in the on-site monkey pox virus typing kit, and further comprises a primer probe for orthopoxvirus genus identification:
forward primer NVAR-F having the nucleotide sequence shown as SEQ ID NO.13, reverse primer NVAR-R having the nucleotide sequence shown as SEQ ID NO.14, and fluorescent-labeled probe NVAR-P-MGB having the nucleotide sequence shown as SEQ ID NO. 15.
NVAR-F:CATCAGACCATATACTGAGTTGGC(SEQ ID NO.13);
NVAR-R:GGCAGAGAGAGCCAGATATAAAAAGA(SEQ ID NO.14);
NVAR-P-MGB:TGGTAGCCTGTTTTAAC(SEQ ID NO.15)。
The probes of the present invention should also include fluorescent reporter groups and quencher groups thereon, as will be generally understood by those skilled in the art. The fluorescent reporter and quencher groups may be in forms that are currently disclosed or not in the art, and serve as labels for PCR detection. The fluorescent reporter group as described may be any one or more of CY3, CY5, CY5.5, FAM, HEX, VIC, JOE, ROX and ATTO 425.
In some specific embodiments, the invention provides specific fluorescent group binding patterns as examples, such as mpxIa-P labeled ATTO425, mpxIb-P labeled FAM, mpxII-P labeled HEX, mpx-P labeled Cy5, NVAR-P-MGB labeled Cy5.
Those skilled in the art will also appreciate that for NVAR-P-MGB, the 3' end may also be ligated to a minor groove binder MGB, in accordance with the present invention. The minor groove binder (Minor Groove Binder, MGB) of the present invention is a dihydropyrrole tripeptide that can selectively bind to minor grooves of DNA molecules, i.e., shallow grooves in DNA helices. The MGB labeled oligonucleotides form stable hybrid complexes with complementary DNA and RNA target sequences.
The actual detection object based on the kit is the monkey pox virus, so that the on-site monkey pox virus typing kit or on-site monkey pox identification and typing kit or on-site monkey pox typing combined orthopoxvirus genus identification kit can be used for controlling the quality of the monkey pox virus drug, and can be realized by verifying the inhibition effect of the monkey pox virus drug on the monkey pox virus. Namely, the invention also provides the on-site monkey pox virus typing kit or the application of the on-site monkey pox virus identification and typing kit or the on-site monkey pox typing combined orthopoxvirus identification kit, wherein the application comprises any one or more of the following:
(1) Used for controlling the quality of monkey pox virus drugs;
(2) Identification of monkey poxvirus;
(3) For monkey poxvirus typing;
(4) For orthopoxvirus identification.
The invention has the beneficial effects that:
(1) A kit for on-site monkey pox virus typing is provided, which can cover the Ib branch which begins to epidemic at the end of 2023 and the II branch which begins epidemic at 2022. Meanwhile, the monkey pox universal target and the orthopoxa target except for smallpox can be detected.
(2) The problem of pathogen rapid detection is solved by utilizing an integrated chip microfluidic mode (as shown in fig. 7), and the sample inlet and the sample outlet are truly realized. The sample was taken and added directly to the apparatus. Integrating nucleic acid extraction, purification, amplification and detection, and obtaining a result in 35 minutes.
(3) The invention optimizes the combination of primer probes to achieve the effect of balanced amplification in the same amplification system, accurately parting the monkey pox virus at one time, and simultaneously identifying the monkey pox virus or orthopoxvirus genus. The kit has the characteristics of high sensitivity and high accuracy, can reduce interference of foreign substances, and has high stability and high practical application value.
Drawings
FIG. 1 shows the results of amplification of the monkey poxvirus Ia branched targets (screen shots).
FIG. 2 shows the amplification results of the monkey poxvirus Ia branch targets (screen shots).
FIG. 3 shows the results of amplification of the branch target of the monkey poxvirus Ib (screen shots).
FIG. 4 shows amplification results of the monkey poxvirus Ib branch target comparative example (screen shots).
FIG. 5 shows the results of amplification of the monkey poxvirus II branched target (screen shots).
FIG. 6 shows the results of amplification of the monkey poxvirus II branched target comparative example (screen shots).
Fig. 7 is a photograph of an integrated microfluidic chip.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Example 1 on-site monkey pox typing kit
The kit of this embodiment comprises the following components:
(1) Primer probe composition
Branching Ia typing primer:
mpxIa-F:ATTATCGATATCTCGAGGCGA(SEQ ID NO.1);
mpxIa-R:AAGAATAGTGTAGAGACTGATGCTAA(SEQ ID NO.2);
mpxIa-P:CATTGATTAAAGAGTGTCCATCCGGTAC(SEQ ID NO.3);
branching Ib typing primer:
mpxIb-F:AGAATTTTCTATTTCAACGGGTATAGC(SEQ ID NO.4);
mpxIb-R:TGGATATGCGCCTGAATATCTAG(SEQ ID NO.5);
mpxIb-P:TGAACACGGCACTTCGAAATGGAAA(SEQ ID NO.6);
Branch II typing primer:
mpxII-F:GTGGTCCCGGAACATATTCT(SEQ ID NO.7);
mpxII-R:GATTCGCTGAGACCGGTAGTG(SEQ ID NO.8);
mpxII-P:TCAACGACACATCGTGTACTCGGACGA(SEQ ID NO.9);
of the above probes mpxIa-P was labeled ATTO425, mpxIb-P was labeled FAM, and mpxII-P was labeled HEX.
(2) Buffer component
The PCR amplification system adopts a commercial system Air-Dryable-Step RT-qPCR Mix, wherein the commercial system Air-Dryable-Step RT-qPCR Mix comprises buffer solution, dNTP Mix, DNA polymerase and UDG.
The air-dried qPCR reaction reagent consists of the components (1) and (2).
(3) Freeze-drying IPC process quality control:
Comprises a freeze-drying system and any one of a positive quality control product and a negative quality control product.
The quality control of the freeze-drying IPC process in this example comprises pseudovirus 1X 10 6 copies/mL, mannitol 10%, trehalose 10%, 2-hydroxypropyl-beta-cyclodextrin HP-beta-CD 0.1%, bovine serum albumin BSA 0.5mg/mL, defoamer SE-15.009%, tris-HCl (pH 8.0) 10mM, naCl 50mM, tween 20.05%.
Namely, the pseudovirus is used as a cationic quality control, wherein the cationic quality control of the pseudovirus comprises target genes of three subtypes of the monkey pox virus, and the anionic quality control of the pseudovirus does not comprise the target genes. The basic process for pseudovirus preparation can be found in chinese patent application No. cn202110669475. X.
In order to ensure the detection accuracy (the quality control product is effective), the kit also comprises a common IPC primer probe composition, and if no special description exists in the detection result, the detection of the quality control product by the IPC primer probe composition is effective, and the IPC primer probe composition specifically comprises:
F:AGTTGCAGTGTAACCGTCATGTA(SEQ ID NO.16);
R:TCGACGAGACTCTGCTGTTAA(SEQ ID NO.17);
P:CAGTAATCTGCGTCGCACGTGTGCA(SEQ ID NO.18)。
example 2 on-site monkey pox identification and typing kit
The difference from example 1 is that:
(1) The primer composition comprises a primer probe for identifying the monkey pox virus besides a parting primer:
mpx-F:TGGAGAAGCGAGAAGTTAATAAAGC(SEQ ID NO.10);
mpx-R:CATCATCTATTATAGCATCAGCATC(SEQ ID NO.11);
mpx-P:CCGTGTATCAGCATCCATTGTCGTAGACC(SEQ ID NO.12);
mpx-P is labeled with Cy5.
The other parts are referred to example 1.
Example 3A Simian poxtyping on site in combination with orthopoxvirus identification assay kit
The difference from example 1 is that:
(1) The primer composition comprises a primer probe for orthopoxvirus detection in addition to a parting primer:
NVAR-F:CATCAGACCATATACTGAGTTGGC(SEQ ID NO.13);
NVAR-R:GGCAGAGAGAGCCAGATATAAAAAGA(SEQ ID NO.14);
NVAR-P-MGB:TGGTAGCCTGTTTTAAC(SEQ ID NO.15)。
NVAR-P-MGB is labeled with Cy5.
The other parts are referred to example 1.
Comparative example 1
The difference between this comparative example and example 1 is that the branched Ia typing primer probe is specifically:
mpx-Ia-F’:TGTCTACCTGGATACAGAAAGCAA;
mpx-Ia-R’:GGCATCTCCGTTTAATACATTGAT;
mpx-Ia-P’:CCCATATATGCTAAATGTACCGGTACCGGA。
comparative example 2
The difference between this comparative example and example 1 is that the branched Ib typing primer probe is different, specifically:
mpx-Ib-F’:AAGACTTCCAAACTTAATCACTCCT;
mpx-Ib-R’:CGTTTGATATAGGATGTGGACATTT;
mpx-Ib-P’:ATATTCAGGCGCATATCCACCCACGT。
comparative example 3
The difference between this comparative example and example 1 is that the branched II typing primer probe is different, specifically:
mpx-II-F:CACACCGTCTCTTCCACAGA;
mpx-II-R:GATACAGGTTAATTTCCACATCG;
mpx-II-P:AACCCGTCGTAACCAGCAATACATTT。
In the embodiment of the present invention, as an example, the whole apparatus used, i.e., the PCR reaction device, is described in chinese patent application No. CN202110057507.0, and the chip apparatus for detecting nucleic acid is described in chinese patent application No. CN 202110055537.8. The amplification procedure used, by way of example, was 55℃for 10min, 95℃for 1min, and 45 cycles of 95℃for 10s,60℃for 20 s.
Effect experimental example 1 method for testing accuracy (specificity) of kit and results thereof
The quality control was tested using the kits of example 1 and comparative examples 1-3, and the positive quality control was diluted to 1.00E+04 copies/mL, respectively, and the results are shown in FIGS. 1-6.
The results show that the positive quality control can be identified in example 1, the amplified Ct values of comparative examples 1-3 are significantly delayed or cannot be amplified, and the Ct value results in FIGS. 1-6 are shown in Table 1.
TABLE 1
Other pathogens were tested using the protocol of example 3, and positive quality controls for detecting specific other pathogens were purchased from the Guangzhou Pond biosciences Inc., with the following information in Table 2:
TABLE 2
Specific amplification results (Ct values) are shown in table 3 below:
TABLE 3 Table 3
From the above data, the specificity of the kit is good.
Effect experiment example 2 kit sensitivity test method and result
The kit of the embodiment is respectively taken for detecting the quality control product, the positive quality control product is respectively diluted to 3200 copies/mL, 1600 copies/mL, 800 copies/mL, 400 copies/mL, 200 copies/mL, 100 copies/mL and 50 copies/mL to be used as samples to be detected, the test is repeated for 10 times, and the detection level of 95% is used as the lowest detection limit of the kit. And counting the Ct value of the lowest detection limit.
The results show that the kits of examples 1-3 can achieve at least 200 copies/mL of identification, and that the results of detecting Ct values (mean ± SD) for each quality control under these conditions are shown in table 4 below, wherein Ia, ib, II are the data of the kit of example 1, mpox are the data of the kit of example 2, and NVAR are the data of the kit of example 3:
TABLE 4 sensitivity test results (lowest detection limit Ct value)
The data show that the kits of examples 1-3 of the invention can be stably distinguished to achieve the identification effect even at the lowest detection limit.
Effect experimental example 3 test method for stability of kit and results
The respective quality control products were measured by using the respective kits of the examples, each positive quality control product was diluted according to the lowest detection limit of each kit (see effect experimental example 2), and after 10 repeated measurements, the CV value of each kit was calculated for evaluation of the measurement stability, and the variation coefficient cv= (standard deviation SD/average Mean) ×100%, the results were as shown in table 5 below, wherein Ia, ib, II are the data of the kit of example 1, mpox are the data of the kit of example 2, and NVAR are the data of the kit of example 3:
Table 5 stability test results (CV value) of the kit
The data show that the kit has good detection stability.
Test method and test result of anti-interference capability of effect experiment example 4 kit
(1) Detection effect on different types of samples
Using the kit of example 1, positive controls were tested 3 times in duplicate using a negative sample of monkey pox virus-free whole blood, vesicles, pustule, and diluted to 200 copies/mL in a gradient,
The statistics of the detected Ct values for different types of samples are shown in table 6 below:
TABLE 6 detection of average Ct values for different sample kits
(2) Detection effect on a Positive reference containing an interferent
For the kit of example 1, the effect of endogenous or exogenous substances possibly present in the sample on the detection result was examined, the cationic control was diluted to 200 copies/mL, bilirubin 500. Mu. Mol/L, triglyceride 10. Mu. Mol/L, azithromycin 100. Mu.g/mL, ribavirin 100. Mu.g/mL were added, and the sample to be tested without interfering substances was used as a reference. Each group was tested for 3 replicates.
The statistical results of the detected Ct values of different interfering substances are shown in table 7 below:
TABLE 7 detection of average Ct values for different interfering substances in kit
The results show that the kit disclosed by the invention is less influenced by different sample types or different interferents, and has higher production value.
The above embodiments are merely for illustrating the technical aspects of the present invention, and do not limit the scope of the present invention. The technical scheme obtained by the technical scheme optimization and adjustment carried out by the person skilled in the art under the condition of not deviating from the technical scheme of the invention is also within the protection scope of the invention. If the simple replacement, replacement and dosage adjustment are carried out on part of components in the amplification system, the obtained scheme adopts the specific primer probe composition of the invention, and the scheme also belongs to the technical scheme of the invention protection, and the scheme is not considered to be different from the invention because the scheme is not presented in the specific embodiment.
Claims (10)
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20060275749A1 (en) * | 2003-12-05 | 2006-12-07 | Isis Pharmaceuticals, Inc. | Compositions for use in identification of orthopoxviruses |
| RU2427648C1 (en) * | 2010-04-30 | 2011-08-27 | Федеральное государственное учреждение науки "Государственный научный центр вирусологии и биотехнологии "Вектор" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека" (ФГУН ГНЦ ВБ "Вектор" Роспотребнадзора) | Set of oligonucleotide primers and fluorescent-marked probes for species-specific instant identification of orthopoxvirus by real-time multiplex pcr |
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| CN116064953A (en) * | 2022-09-29 | 2023-05-05 | 中元汇吉生物技术股份有限公司 | Kit, method and application for detecting and typing nucleic acid of monkey poxvirus in same tube |
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| WO2024148013A1 (en) * | 2023-01-02 | 2024-07-11 | Life Technologies Corporation | Compositions, kits and methods for detection of viral sequences |
| CN119753228A (en) * | 2024-11-14 | 2025-04-04 | 深圳普门科技股份有限公司 | Primer probe combination and kit for detecting orthopoxvirus and detecting monkey poxvirus and typing |
| CN120249558A (en) * | 2025-03-30 | 2025-07-04 | 昆明理工大学 | A primer combination for detecting monkeypox virus and distinguishing monkeypox virus types Ⅰa, Ⅰb, and Ⅱ and its application |
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2025
- 2025-07-31 CN CN202511062843.9A patent/CN120536642A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060275749A1 (en) * | 2003-12-05 | 2006-12-07 | Isis Pharmaceuticals, Inc. | Compositions for use in identification of orthopoxviruses |
| RU2427648C1 (en) * | 2010-04-30 | 2011-08-27 | Федеральное государственное учреждение науки "Государственный научный центр вирусологии и биотехнологии "Вектор" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека" (ФГУН ГНЦ ВБ "Вектор" Роспотребнадзора) | Set of oligonucleotide primers and fluorescent-marked probes for species-specific instant identification of orthopoxvirus by real-time multiplex pcr |
| CN114752711A (en) * | 2022-06-15 | 2022-07-15 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting and parting monkeypox virus and application thereof |
| CN115466800A (en) * | 2022-06-20 | 2022-12-13 | 浙江大学医学院附属第一医院 | A multiplex fluorescent quantitative PCR kit for detecting monkeypox virus and its subtypes |
| CN116064953A (en) * | 2022-09-29 | 2023-05-05 | 中元汇吉生物技术股份有限公司 | Kit, method and application for detecting and typing nucleic acid of monkey poxvirus in same tube |
| CN116219071A (en) * | 2022-12-14 | 2023-06-06 | 广州凯普医药科技有限公司 | Multiplex qPCR kit for simultaneously detecting and identifying type I, type IIa and type IIb monkey poxviruses |
| WO2024148013A1 (en) * | 2023-01-02 | 2024-07-11 | Life Technologies Corporation | Compositions, kits and methods for detection of viral sequences |
| CN119753228A (en) * | 2024-11-14 | 2025-04-04 | 深圳普门科技股份有限公司 | Primer probe combination and kit for detecting orthopoxvirus and detecting monkey poxvirus and typing |
| CN120249558A (en) * | 2025-03-30 | 2025-07-04 | 昆明理工大学 | A primer combination for detecting monkeypox virus and distinguishing monkeypox virus types Ⅰa, Ⅰb, and Ⅱ and its application |
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