Primer pair, probe, kit and detection method for norovirus detection

Abstract

The invention belongs to the field of in-vitro diagnosis and detection, and particularly relates to a primer pair, a probe, a kit and a detection method for norovirus detection. The norovirus detection primer pair provided by the invention comprises a primer pair for detecting a norovirus GI type, a primer pair for detecting a norovirus GII type and a primer pair for detecting a norovirus GIV type, and specific primers and probes are designed on the basis of a norovirus GI / GII / GIV type gene conserved region; and a plurality of pairs of primers or probes are introduced, and degenerate primers and degenerate probes are introduced, so that the existing norovirus subtypes are better matched, the detection coverage rate is increased, and the omission ratio is reduced.

Description

Technical Field

[0001] The present application relates to the field of in vitro diagnostic testing, and specifically relates to primer pairs, probes, kits and detection methods for norovirus detection. Background Art

[0002] Norovirus belongs to the genus Norovirus (NV) of the family Human Caliciviridae (HuCV). It is a nonenveloped, single-stranded, positive-sense RNA virus with a virion diameter of approximately 27–40 nm and a genome length of approximately 7.5–7.7 kb. It consists of three open reading frames (ORFs), flanked by 5' and 3' untranslated regions (UTRs) and a polyadenylated tail (PolyA) at the 3' end. ORF1 encodes a polyprotein that is cleaved into six non-structural polyproteins involved in replication after translation. ORF2 and ORF3 encode the major structural protein (VP1) and minor structural protein (VP2), respectively. Based on genomic characteristics and phylogenetic analysis, noroviruses have been divided into at least six genogroups (GⅠ–GⅥ). Among them, GI and GII are the two main gene groups that cause acute gastroenteritis in humans. GIV can also infect humans but is rarely detected, while GIII, GV and GVI infect cattle, mice and dogs respectively.

[0003] Norovirus is a zoonotic pathogen that causes severe diarrhea and is also a significant cause of foodborne illness. Norovirus can be transmitted from person to person, through food, and through water. Norovirus has diverse transmission routes, a low infectious dose, a long detoxification period, strong environmental resistance, rapid viral mutation, and a short duration of immune protection. It is highly contagious, spreads rapidly, and is prone to mutation and recombination.

[0004] Norovirus cannot be cultured in vitro, serotyped, or identified. There are no effective treatments or vaccines, requiring only rapid early detection for risk screening. Currently, norovirus identification is primarily performed through electron microscopy, immunology, or molecular biology. While electron microscopy allows direct visualization of norovirus particles, it is complex, time-consuming, and tedious, with high subjectivity and low sensitivity. It often struggles to detect small amounts of virus, and requires expensive instrumentation and experienced operators, limiting its practical application. While immunology methods offer shorter detection times than electron microscopy, they still have a detection window, poor sensitivity, and are prone to cross-reactivity when antibodies are less specific. Furthermore, immunology testing poses biosafety risks. Molecular biology methods are the primary method for clinical norovirus detection due to their high sensitivity, specificity, and accuracy, along with their simplicity, short detection time, and fully contained procedures that prevent specimen contamination and environmental pollution. Molecular biology methods have become the primary method for clinical norovirus detection.

[0005] At present, fluorescent PCR kits for norovirus mainly detect GI type and GII type. However, the transmission mode of norovirus of different genotypes is different. Timely acquisition of norovirus typing information can assist in inferring its transmission route and help prevent and control the epidemic. At present, the typing of norovirus is mainly based on whole genome sequencing, which is time-consuming and expensive. Patent CN110273027A discloses a nucleic acid typing detection kit and detection method for norovirus GI, GII and GIV types. However, the detection time of the kit is long and the published primer and probe sequences are poorly conserved, which directly leads to poor detection tolerance of the reagent and easy missed detection; Patent CN113930545A discloses a norovirus nucleic acid detection kit and its use method, which can simultaneously detect infection of norovirus GI type and GII type, but cannot detect GIV type; Patent CN112760423A uses LAMP technology to quickly detect norovirus GI type and GII type in food, but it is easy to cause aerosol contamination and affect the detection results.

[0006] In summary, traditional technologies for detecting norovirus are prone to missed detection and have problems with low sensitivity and specificity. Summary of the Invention

[0007] Based on this, an embodiment of the present application provides a primer pair for norovirus detection. Norovirus detection using this primer pair has high sensitivity and specificity, and is not prone to missed detection.

[0008] On the one hand, the present application provides a norovirus detection primer pair, wherein the detection primer pair is selected from one or more of the following primer pairs:

[0009] A primer pair for detecting norovirus GI type, comprising: an upstream primer having a sequence as shown in SEQ ID NO.1 to SEQ ID NO.4, and a downstream primer having a sequence as shown in SEQ ID NO.5;

[0010] A detection primer pair for detecting norovirus GII type, comprising: an upstream primer having a sequence as shown in SEQ ID NO.15 to SEQ ID NO.17, and a downstream primer having a sequence as shown in SEQ ID NO.18; and

[0011] The detection primer pair for detecting norovirus GIV type includes: an upstream primer with a sequence as shown in SEQ ID NO.30, and a downstream primer with a sequence as shown in SEQ ID NO.31.

[0012] On the other hand, the present application provides a norovirus detection probe, which can specifically bind to the amplified fragment of the detection primer pair according to claim 1;

[0013] Optionally, the detection probe comprises:

[0014] The GI-type probe has a sequence as shown in SEQ ID NO.6;

[0015] GII type probes whose sequences are shown in SEQ ID NO.19 to SEQ ID NO.20;

[0016] The sequence of the GIV type probe is shown in SEQ ID NO.32.

[0017] In one embodiment, the detection probes are each independently labeled with a fluorescent reporter group and a fluorescent quencher group, and the fluorescent reporter groups labeled with each detection probe are different.

[0018] On the other hand, the present application provides a norovirus detection kit, characterized in that the norovirus detection kit comprises the detection primer pair and / or the detection probe;

[0019] Optionally, the detection kit further comprises an internal reference primer pair and an internal reference probe for detecting an internal reference gene.

[0020] In one embodiment, the internal reference primer pair includes an upstream primer whose sequence is shown in SEQ ID NO.41 and a downstream primer whose sequence is shown in SEQ ID NO.42; the internal reference probe sequence includes an internal reference probe shown in SEQ ID NO.43.

[0021] In one embodiment, the detection kit further includes one or more of a norovirus nucleic acid extract, a norovirus PCR reaction solution, and a norovirus PCR enzyme solution.

[0022] In one embodiment, the norovirus nucleic acid extract includes 80mM~120mMTris-HCl buffer with a pH of 8.2~8.4, 1.0%~1.4% (v / v) NP-40, 1.8~2.2% (v / v) TritonX-100, 2.4%~2.6% (v / v) Tween-80, 80mM~120mM KCl, 28mM~32mM NaOH, 0.8U / μL~1.2U / μL RNase inhibitor and 120mg / mL~130mg / mL nanomagnetic beads.

[0023] In one embodiment, the norovirus PCR reaction solution includes a pH of 8.8-9.2, 48 mM-52 mM Tris buffer, 80 mM-120 mM potassium chloride, 3.8 mM-4.2 mM magnesium sulfate, 0.6%-0.8% (v / v) Triton X-100, 0.4 mg / mL-0.8 mg / mL bovine serum albumin, 0.08 U / μL-0.12 U / μL inorganic pyrophosphatase, 4.8%-5.2% (v / v) DMSO, and 0.18 mM-0.22 mM dNTPs; and / or

[0024] The norovirus PCR enzyme solution includes 0.50U / μL to 0.60U / μL reverse transcriptase, 0.12U / μL to 0.13U / μL Taq DNA polymerase, 0.8U / μL to 1.2U / μL RNasin, and 0.08U / μL to 0.12U / μL Heat-UDG.

[0025] In one embodiment, the detection kit further includes one or both of a negative control and a positive control;

[0026] Optionally, the negative control includes physiological saline; the positive control includes a pseudovirus containing norovirus GI type, a pseudovirus containing norovirus GII type, a pseudovirus containing norovirus GIV type, and a pseudovirus containing an internal reference gene specific fragment.

[0027] On the other hand, the present application provides a method for typing and detecting norovirus, characterized by comprising:

[0028] The norovirus nucleic acid to be tested is extracted from the sample, and the fluorescent quantitative PCR reaction is performed using the norovirus detection kit to obtain an amplified product; and the fluorescent signal is collected to type the norovirus.

[0029] In one embodiment, the sample is a human stool sample or a human vomitus sample;

[0030] In one embodiment, the fluorescent quantitative PCR reaction is a one-step multiplex fluorescent quantitative PCR reaction;

[0031] In one embodiment, the amplification program of the PCR reaction includes: 54°C to 56°C, reaction for 1 min to 3 min; 94°C to 96°C, reaction for 0.5 min to 1.5 min; 93°C to 95°C, reaction for 0.8s to 1.2s, 55°C to 65°C, reaction for 12s to 14s, for a total of 5 cycles; 90°C to 94°C, reaction for 0.8s to 1.2s, 55°C to 65°C, reaction for 12s to 14s, collecting fluorescence signals, for a total of 40 cycles.

[0032] The details of one or more embodiments of the present application are set forth in the description below, and other features, objects, and advantages of the application will become apparent from the description and from the claims thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0034] Figure 1 For the screening of primers and probes for Norovirus GI type;

[0035] Figure 2 For the screening of primers and probes for Norovirus GII type;

[0036] Figure 3 For the screening of primers and probes for Norovirus GIV type;

[0037] Figure 4 Sensitivity analysis of the detection kit provided in one embodiment of the present application;

[0038] Figure 5 This is a specific analysis of the detection kit provided in one embodiment of the present application. DETAILED DESCRIPTION

[0039] Below in conjunction with embodiment and example, the application is described in further detail.Should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description hereinafter, a large amount of specific details are given in order to provide a more complete understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0041] the term

[0042] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0043] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0044] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0045] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0046] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0047] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0048] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0049] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0050] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0051] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0052] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0053] All documents mentioned in this application are cited as references in this application, just as each document is cited as a reference individually. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0054] The present application provides a one-step multiplex fluorescence quantitative PCR detection kit with high sensitivity, strong specificity, high accuracy, strong inclusiveness and accurate typing. It can accurately type GⅠ, GⅡ and GⅣ types in the same reaction system, which can greatly shorten the detection time and conduct risk screening through rapid detection in the early stage of the epidemic, which is conducive to the timely confirmation and control of the norovirus epidemic and is suitable for the promotion and application of large-scale screening and diagnosis.

[0055] On the one hand, the present application provides a norovirus detection primer pair, wherein the detection primer pair is selected from one or more of the following primer pairs:

[0056] A primer pair for detecting norovirus GI type, comprising: an upstream primer having a sequence as shown in SEQ ID NO.1 to SEQ ID NO.4, and a downstream primer having a sequence as shown in SEQ ID NO.5;

[0057] A detection primer pair for detecting norovirus GII type, comprising: an upstream primer having a sequence as shown in SEQ ID NO.15 to SEQ ID NO.17, and a downstream primer having a sequence as shown in SEQ ID NO.18; and

[0058] The detection primer pair for detecting norovirus GIV type includes: an upstream primer with a sequence as shown in SEQ ID NO.30, and a downstream primer with a sequence as shown in SEQ ID NO.31.

[0059] Specifically, they include:

[0060] (1) GI type primer sequence

[0061] GI-F1-1:5'-AT G TT C CG C TGGATGCG-3' (SEQ ID NO. 1)

[0062] GI-R1: 5'-CCTTAGACGCCATCATCATTTAC-3' (SEQ ID NO.5)

[0063] At the same time, considering that GI-F1-1 has a 3-base mismatch with other mutant GI-type norovirus genes, GI-F1-2: 5'-ATGTTCCG was also specifically designed. T TGGATGCG-3' (SEQ ID NO. 2)

[0064] GI-F1-3:5'-AT A TTCCGCTGGATGCG-3'(SEQ ID NO.3)

[0065] GI-F1-4:5'-ATGTT T CGCTGGATGCG-3' (SEQ ID NO. 4)

[0066] The above four pairs of primers can be effectively used simultaneously by being paired with GI-R1 respectively to amplify as many GI-type norovirus variants as possible.

[0067] (2) GII type primer sequence

[0068] GII-F1-1 is a degenerate primer: 5'-ATGTT C AGRTGGATGAGRTT C TC-3' (SEQ ID NO. 15), wherein R represents an A or G base;

[0069] GII-R1: 5'-TCGACGCCATCTTCATTCACA-3' (SEQ ID NO. 18). At the same time, considering that GII-F1-1 has mismatched bases with other GII-type norovirus variants, in addition to using the R degenerate base, GII-F1-2: 5'-ATGTTCAGGTGGATGAGGTT was also specifically designed. T TC-3' (SEQ ID NO. 16)

[0070] GII-F1-3:5'-ATGTT T AGGTGGATGAGATTCTC-3' (SEQ ID NO. 17) was paired with GII-R1 to amplify GII type norovirus; the above three pairs of primers can be effectively used simultaneously to amplify as many GII type norovirus variants as possible.

[0071] (3) GIV type primer sequence

[0072] The primer pair for detecting norovirus GIV type includes: an upstream primer whose sequence is shown in SEQ ID NO.30, and a downstream primer whose sequence is shown in SEQ ID NO.31:

[0073] GIV-F1: 5'-TCGACGCCATCTTCATTCACA-3' (SEQ ID NO.30)

[0074] GIV-R1: 5'-GAGTCCATGTACAAGTGGATGC-3' (SEQ ID NO.31)

[0075] On the other hand, the present application provides a norovirus detection probe, which can specifically bind to the amplified fragment of the detection primer pair;

[0076] The detection probe is selected from one or more of the following detection probes:

[0077] (1) GI type probe sequence

[0078] GI-P1 is a degenerate probe: 5'-FAM-TGTGGACAGGAGAYCGCRATCT-BHQ1-3', where Y in the sequence represents a C or T base, and R represents an A or G base.

[0079] The GI type probe sequence is shown in SEQ ID NO.6:

[0080] GI-P1: FAM-TGTGGACAGGAGAYCGCRATCT-BHQ1 (SEQ ID NO.6)

[0081] (2) GII type probe sequence

[0082] GII-P1-1:5'-VIC-AGCAC G TGGGAGGGCGATCG-BHQ1-3'. At the same time, in order to detect as many GII noroviruses as possible, an additional specific probe GII-P1-2: 5'-VIC-AGCAC was designed. A TGGGAGGGCGATCG-BHQ1-3'; used to detect GII norovirus variants.

[0083] The GII type probe sequences are shown in SEQ ID NO.19 to SEQ ID NO.20:

[0084] GII-P1-1: VIC-AGCAC G TGGGAGGGCGATCG-BHQ1(SEQ ID NO.19)

[0085] GII-P1-2: VIC-AGCAC A TGGGAGGGCGATCG-BHQ1(SEQ ID NO.20)

[0086] (3) GIV probe sequence

[0087] GIV-P1 is a degenerate probe: 5'-CY5-AATCGGGAGCRAGATCGCGATCSCCCTC-BHQ2-3', where R represents A or G base and S represents C or G.

[0088] The sequence of the GIV type probe is shown in SEQ ID NO.32:

[0089] GIV-P1: CY5-AATCGGGAGCRAGATCGCGATCSCCCTC-BHQ2 (SEQ ID NO. 32).

[0090] In a specific example, the detection probes are each independently labeled with a fluorescent reporter group and a fluorescent quencher group, and the fluorescent reporter groups labeled with each detection probe are different.

[0091] The primer-probe mixture for norovirus GI, GII and GIV genotyping includes a primer pair for detecting norovirus GI type and a fluorescent probe with a 5' end labeled with a reporter fluorescent dye FAM and a 3' end labeled with a quenching fluorescent dye BHQ1; a primer pair for detecting norovirus GII type and a probe with a 5' end labeled with a reporter fluorescent dye VIC or HEX and a 3' end labeled with a quenching fluorescent dye BHQ1; a primer pair for detecting norovirus GIV type and a probe with a 5' end labeled with a reporter fluorescent dye CY5 and a 3' end labeled with a quenching fluorescent dye BHQ2.

[0092] This application designs specific primers and probes for the conserved regions of the norovirus GI / GII / GIV genes. By introducing multiple pairs of primers or probes, as well as degenerate primers or probes, this ensures better matching of existing norovirus subtypes, improves detection coverage, and reduces missed detection rates. This maximizes coverage of the existing published norovirus GI, GII, and GIV genotypes while avoiding cross-reactions with other subtypes of norovirus or other microorganisms, thereby improving detection coverage and reducing missed detection rates.

[0093] On the other hand, the present application provides a norovirus detection kit, which includes the detection primer pair and / or the detection probe, as well as an internal reference primer pair and an internal reference probe for detecting an internal reference gene.

[0094] In a specific example, the internal reference primer pair includes an upstream primer whose sequence is shown in SEQ ID NO.41 and a downstream primer whose sequence is shown in SEQ ID NO.42; the internal reference probe sequence includes an internal reference probe shown in SEQ ID NO.43.

[0095] Internal reference upstream primer IC-F: 5′-AGATTTGGACCTGCGAGCG-3′ (SEQ ID NO. 41);

[0096] Internal reference downstream primer IC-R: 5′-GAGCGGCTGTCTCCACAAGT-3′ (SEQ ID NO. 42);

[0097] Internal control probe IC-P: ROX-TTCTGACCTGAAGGCTCTGCGCG-BHQ2 (SEQ ID NO. 43).

[0098] By setting up endogenous internal references, this application can monitor the entire process of sample collection, storage and transportation, as well as nucleic acid extraction and amplification, to avoid the occurrence of false negative results, and thus determine the authenticity of the test results.

[0099] Optionally, the kit further includes one or more of a norovirus nucleic acid extract, a norovirus PCR reaction solution, and a norovirus PCR enzyme solution.

[0100] In a specific example, the norovirus nucleic acid extract includes 80mM~120mMTris-HCl buffer with a pH of 8.2~8.4, 1.0%~1.4% (v / v) NP-40, 1.8~2.2% (v / v) TritonX-100, 2.4%~2.6% (v / v) Tween-80, 80mM~120mM KCl, 28mM~32mM NaOH, 0.8U / μL~1.2U / μL RNase inhibitor and 120mg / mL~130mg / mL nanomagnetic beads.

[0101] The nucleic acid extraction solution used in this application is an independently designed method that innovatively combines a one-step nucleic acid releaser with nanomagnetic beads. This method not only rapidly releases nucleic acids within 5 minutes at room temperature, but also allows for nucleic acid purification using magnetic beads, shortening extraction time and improving efficiency. It also reduces the inhibition of PCR reactions by interfering substances in the sample. Furthermore, all extracted nucleic acids participate in the PCR amplification process, preventing RNA loss and effectively improving detection sensitivity.

[0102] In a specific example, the norovirus PCR reaction solution includes a pH of 8.8 to 9.2, 48 mM to 52 mM Tris buffer, 80 mM to 120 mM potassium chloride, 3.8 mM to 4.2 mM magnesium sulfate, 0.6% to 0.8% (v / v) Triton X-100, 0.4 mg / mL to 0.8 mg / mL bovine serum albumin, 0.08 U / μL to 0.12 U / μL inorganic pyrophosphatase, 4.8% to 5.2% (v / v) DMSO, and 0.18 mM to 0.22 mM dNTPs.

[0103] In a specific example, the norovirus PCR enzyme solution includes 0.50U / μL to 0.60U / μL reverse transcriptase, 0.12U / μL to 0.13U / μL Taq DNA polymerase, 0.8U / μL to 1.2U / μL RNasin, and 0.08U / μL to 0.12U / μL Heat-UDG.

[0104] This application adopts the design of dUTP+thermosensitive UDG enzyme anti-contamination system, which can eliminate the contamination of PCR products containing uracil, effectively prevent aerosol contamination caused by PCR amplification products, reduce false positives caused by amplification product contamination, and improve the accuracy of reagents.

[0105] Optionally, the kit further comprises one or both of a negative control and a positive control.

[0106] Further optionally, the negative control includes physiological saline, and the positive control includes a pseudovirus containing norovirus GI type, norovirus GII type, norovirus GIV type and an internal reference gene specific fragment.

[0107] Another aspect of the present application provides a method for detecting norovirus typing, comprising:

[0108] Extracting the norovirus nucleic acid to be tested from the sample, performing a multiplex fluorescence quantitative PCR reaction using the norovirus detection kit to obtain an amplified product; and

[0109] Collect fluorescence signals and type norovirus.

[0110] For example: select the FAM channel to detect GI type norovirus target genes; select the VIC / HEX channel to detect GII type norovirus target genes; select the CY5 channel to detect GIV type norovirus target genes; select the ROX channel to detect internal reference genes.

[0111] During detection, threshold setting: manually adjust the threshold line so that the threshold line just exceeds the highest point of the negative control amplification curve (irregular noise line).

[0112] Interpretation of results:

[0113] (1) Internal reference (ROX) Ct≤35:

[0114] 1.1 If the Ct value of the FAM channel is ≤35, and the Ct values ​​of the VIC and CY5 channels are both >35 or no Ct value is found, then the test result is positive for Norovirus GI type, and negative for Norovirus GII and GIV types.

[0115] 1.2 If the Ct value of the VIC channel is ≤35, and the Ct values ​​of the FAM and CY5 channels are both >35 or no Ct value is found, then the test result is positive for Norovirus GII, and negative for Norovirus G1 and GIV.

[0116] 1.3 If the Ct value of the CY5 channel is ≤ 35, and the Ct values ​​of the FAM and VIC channels are both > 35 or no Ct value is found, then the test result is positive for norovirus GIV and negative for norovirus GI and GII.

[0117] 1.4 If the Ct values ​​of the FAM and VIC channels are both ≤35, and the Ct value of the CY5 channel is >35 or no Ct value is found, then the test is positive for norovirus GI and GII types, and negative for norovirus GIV type;

[0118] 1.5 If the Ct values ​​of the FAM and CY5 channels are both ≤35, and the Ct value of the VIC channel is >35 or no Ct value is found, then the test is positive for norovirus GI and GIV types, and negative for norovirus GII type;

[0119] 1.6 If the Ct values ​​of the VIC and CY5 channels are both ≤35, and the Ct value of the FAM channel is >35 or no Ct value is found, then the test is positive for norovirus GII and GIV, and negative for norovirus GI.

[0120] 1.7 If the Ct values ​​of the FAM, VIC, and CY5 channels are all ≤35, then the Norovirus GI, GII, and GIV types are all positive;

[0121] 1.8 If the Ct values ​​for the FAM, VIC, and CY5 channels are all greater than 35 or there is no Ct value, then the Norovirus GI, GII, and GIV types are all negative.

[0122] (2) If the internal reference (ROX channel) Ct value is greater than 35 or there is no Ct value, the test is invalid and needs to be repeated.

[0123] In one specific example, the sample includes a human stool sample or a human vomitus sample;

[0124] In a specific example, the multiplex fluorescence quantitative PCR reaction is a one-step multiplex fluorescence quantitative PCR reaction;

[0125] This application adopts one-step multiplex fluorescence quantitative PCR technology. By adding multi-channel primer probes specific for norovirus GⅠ, GⅡ and GⅣ types into the same PCR reaction system, reverse transcription and PCR amplification are completed in one tube, achieving sensitive detection and accurate typing of GI / GII / GIV types. The PCR amplification process can be completed within 40 minutes, which is conducive to the rapid screening of large numbers of clinical samples.

[0126] In a specific example, the amplification program of the PCR reaction includes: 54℃~56℃, reaction 1min~3min; 94℃~96℃, reaction 0.5min~1.5min; 93℃~95℃, reaction 0.8s~1.2s, 55℃~65℃, reaction 12s~14s, for a total of 5 cycles; 90℃~94℃, reaction 0.8s~1.2s, 55℃~65℃, reaction 12s~14s, collecting fluorescence signals, for a total of 40 cycles.

[0127] The present application provides a primer pair for detecting norovirus, including a primer pair for detecting norovirus GI type, a primer pair for detecting norovirus GII type, and a primer pair for detecting norovirus GIV type. The present application is based on the design of specific primers and probes for the conserved regions of the norovirus GI / GII / GIV type genes, and by introducing multiple pairs of primers or probes, degenerate primers, and degenerate probes, ensures better matching of existing norovirus subtypes, improves detection coverage, and reduces missed detection rates.

[0128] Furthermore, the present application adopts rapid nucleic acid extraction and one-step multiplex fluorescence quantitative PCR technology to complete reverse transcription and PCR amplification in one tube, realizing sensitive detection and accurate typing of GI / GII / GIV types, and can complete the PCR amplification process within 40 minutes, which is conducive to the rapid screening of large numbers of clinical samples.

[0129] This application also optimizes the nucleic acid extraction time and PCR system, and adopts a one-step nucleic acid releaser combined with nanomagnetic beads. It can quickly release nucleic acids within 5 minutes under normal temperature conditions, and can also use magnetic beads for nucleic acid purification, shortening the extraction time and improving the extraction efficiency. It can also reduce the inhibition of the PCR reaction by related interfering substances in the sample. And all the extracted nucleic acids participate in the PCR amplification process, which will not cause the loss of RNA and can effectively improve the detection sensitivity. And the design of the dUTP+thermosensitive UDG enzyme anti-pollution system can eliminate the contamination of PCR products containing uracil, effectively prevent aerosol contamination caused by PCR amplification products, reduce false positives caused by amplification product contamination, and further improve the accuracy of the reagents.

[0130] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0131] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0132] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0133] Example 1

[0134] 1. Screening of primers and probes

[0135] This example is based on the published sequence information of norovirus GⅠ, GⅡ and GIV genotypes. The published gene sequences of norovirus GⅠ, GⅡ and GIV were downloaded from the NCBI virus database and compared and analyzed. The candidate primers and probes shown in Table 1, Table 2 and Table 3 were designed in their conserved regions. All primers and probes were synthesized by Shanghai Bioengineering. The plasmids of GI / GII / GIV types were diluted in nuclease-free double distilled water from 1 ng / μL to 10 -1 ng / μL, 10 -2 ng / μL, 10 -3 ng / μL, 10 -4 ng / μL, 10 -5 ng / μL, 10 -6 ng / μL, 10 -7 ng / μL, 10 -8 ng / μL, the reaction system was prepared using the commercial premix (2×HS Taq One-step RT PCR Master Mix-3 (Plus1), catalog number MDAR041) produced by Feipeng Bio according to the instructions, and the amplification efficiency of the candidate primers and probes was analyzed on a BioRad CFX Opus96 fluorescence quantitative PCR instrument.

[0136] Table 1: GI-type primer probe sequences

[0137]

[0138]

[0139] Table 2: GII type primer probe sequences

[0140]

[0141]

[0142] Table 3 GIV type primer probe sequences

[0143]

[0144] The results of the primer and probe screening study for Norovirus GI, GII, and GIV are as follows: Figure 1 、 Figure 2 、 Figure 3 As shown, the primer-probe amplification efficiency of GI type combination 1, GII type combination 4, and GIV type combination 7 is higher. Combinations 1, 4, and 7 will be used as primer probes for detecting norovirus GI, GII, and GIV types in the future. The internal reference primers and probes are shown in Table 4 below:

[0145] Table 4: Internal reference primers and probes

[0146]

[0147] 2. Preparation of Norovirus GI, GII and GIV Type Nucleic Acid Typing Fluorescence PCR Detection Kit

[0148] The specific components of the Norovirus GI, GII and GIV type nucleic acid typing fluorescent PCR detection kit include: Norovirus nucleic acid extract, NV (Norovirus) PCR reaction solution, NV (Norovirus) PCR enzyme solution, positive control and negative control.

[0149] The preparation method of norovirus nucleic acid extract is as follows: Tris-HCl buffer with a pH of 8.3, NP-40, TritonX-100, Tween-80, KCl, NaOH, and RNase inhibitor are mixed in a certain proportion, filtered, sterilized, and impurities removed, and a certain amount of nanomagnetic beads are added to prepare the norovirus nucleic acid extract. The specific preparation method is shown in Table 5, the NV PCR reaction solution is shown in Table 6, and the NV PCR enzyme solution formula is shown in Table 7.

[0150] Table 5: Preparation method of Norovirus nucleic acid extract

[0151] Serial number Material Name concentration 1 Tris-HCl buffer (pH 8.3) 100mM 2 NP-40 1.2％ 3 TritonX-100 2％ 4 Tween-80 2.5％ 5 KCl 100mM 6 NaOH 30mM 7 RNase inhibitors 1U / μL 8 Nanomagnetic beads 125 mg / mL 9 Nuclease-free double-distilled water Make up the volume

[0152] The pseudovirus containing GI, GII, GIV and internal reference gene specific fragments was diluted with physiological saline to 1×106 The positive control of this kit is 1000 copies / mL. The negative control is normal saline.

[0153] Table 6: NV PCR reaction solution preparation method

[0154]

[0155]

[0156] Table 7: NV PCR enzyme solution preparation method

[0157] Serial number Material Name Final concentration 1 Reverse transcriptase 0.55 U / μL 2 Taq DNA polymerase 0.125 U / μL 3 RNase inhibitors 1U / μL 4 Heat-UDG 0.1 U / μL

[0158] 3. Detection method of the Norovirus GⅠ, GⅡ and GⅣ nucleic acid typing fluorescent PCR detection kit. The detection method of the Norovirus GⅠ, GⅡ and GⅣ nucleic acid typing fluorescent PCR detection kit comprises the following steps:

[0159] (1) Reagent Preparation: Thaw the NV PCR reaction solution and NV PCR enzyme solution at room temperature, vortex thoroughly to mix, and centrifuge briefly to prepare the NV PCR-mix. Add 23 μL of NV PCR reaction solution and 2 μL of NV PCR enzyme solution per reaction. Transfer the prepared PCR-mix to the sample processing area.

[0160] (2) Nucleic acid extraction and sample addition: Take 200 μL of the sample to be tested and 200 μL of norovirus nucleic acid extract and mix them in equal volumes. Let them stand at room temperature for 5 minutes. After standing, carefully remove the upper waste liquid. Transfer all the magnetic beads adsorbed with nucleic acid in the lower layer to a fluorescent quantitative PCR tube containing 25 μL PCR-mix. After loading the tube, follow the PCR amplification program in Table 8 in ABIQuantStudio. TM 5. Perform amplification on a real-time fluorescence quantitative PCR instrument.

[0161] Table 8: PCR amplification program

[0162]

[0163] Instrument channel selection: Select the FAM channel to detect GI type norovirus target genes; select the VIC / HEX channel to detect GII type norovirus target genes; select the CY5 channel to detect GIV type norovirus target genes; select the ROX channel to detect internal reference genes.

[0164] Threshold setting: Manually adjust the threshold line so that it just exceeds the highest point of the negative control amplification curve (irregular noise line).

[0165] Interpretation of results:

[0166] 1) Internal reference (ROX) Ct≤35

[0167] 1.1 If the Ct value of the FAM channel is ≤35, and the Ct values ​​of the VIC and CY5 channels are both >35 or no Ct value is found, then the test result is positive for Norovirus GI type, and negative for Norovirus GII and GIV types.

[0168] 1.2 If the Ct value of the VIC channel is ≤35, and the Ct values ​​of the FAM and CY5 channels are both >35 or no Ct value is found, then the test result is positive for Norovirus GII, and negative for Norovirus G1 and GIV.

[0169] 1.3 If the Ct value of the CY5 channel is ≤ 35, and the Ct values ​​of the FAM and VIC channels are both > 35 or no Ct value is found, then the test result is positive for norovirus GIV and negative for norovirus GI and GII.

[0170] 1.4 If the Ct values ​​of the FAM and VIC channels are both ≤35, and the Ct value of the CY5 channel is >35 or no Ct value is found, then the test is positive for norovirus GI and GII types, and negative for norovirus GIV type;

[0171] 1.5 If the Ct values ​​of the FAM and CY5 channels are both ≤35, and the Ct value of the VIC channel is >35 or no Ct value is found, then the test is positive for norovirus GI and GIV types, and negative for norovirus GII type;

[0172] 1.6 If the Ct values ​​of the VIC and CY5 channels are both ≤35, and the Ct value of the FAM channel is >35 or no Ct value is found, then the test is positive for norovirus GII and GIV, and negative for norovirus GI.

[0173] 1.7 If the Ct values ​​of the FAM, VIC, and CY5 channels are all ≤35, then the Norovirus GI, GII, and GIV types are all positive;

[0174] 1.8 If the Ct values ​​for the FAM, VIC, and CY5 channels are all greater than 35 or there is no Ct value, then the Norovirus GI, GII, and GIV types are all negative.

[0175] 2) If the internal reference (ROX channel) Ct value is greater than 35 or there is no Ct value, the test is invalid and needs to be repeated.

[0176] 4. Performance Analysis

[0177] 1. Sensitivity analysis

[0178] After the artificially synthesized GI, GII and GIV pseudoviruses were determined by digital PCR, their concentrations were diluted to 3×10 6 The three pseudoviruses were mixed in equal volumes and diluted sequentially with nuclease-free double-distilled water to 1×103 The concentrations of the test kit were selected from the group consisting of copies / mL, 500 copies / mL, 250 copies / mL, 200 copies / mL, 150 copies / mL, 100 copies / mL, 50 copies / mL, and 25 copies / mL. After nucleic acid extraction, the norovirus nucleic acid extract in the kit of this application was used, and the test was repeated 20 times. The concentration with a detection rate of more than 95% was used as the sensitivity of the kit of this application.

[0179] The analysis results are shown in Figure 4 The results showed that this kit can effectively 3 The detection rates for pseudoviruses of GI, GII, and GIV at concentrations of 100 copies / mL, 500 copies / mL, 250 copies / mL, 200 copies / mL, and 150 copies / mL were all 100%. The detection rates for pseudoviruses of GI, GII, and GIV at 100 copies / mL were all 95%. However, the detection rates for pseudoviruses of GI, GII, and GIV at concentrations of 50 copies / mL and 25 copies / mL were all below 95%. Therefore, the final sensitivity of this kit was determined to be 100 copies / mL.

[0180] 2. Specificity analysis

[0181] Using the norovirus nucleic acid extract and detection kit provided by this application, 22 virus quality control products or bacterial quality control products and the NV positive control of the kit were cross-reacted according to the detection method of Example 3. The specific detection results are as follows Figure 5 As shown. This kit detects NV positive control, GI / GII / GIV / internal reference results are all positive, and other viruses or bacteria are all norovirus negative. It shows that the kit of the present invention does not cross-react with quality control products such as herpes simplex virus type 1 / 2, enteric adenovirus, varicella-zoster virus, rotavirus, hepatitis A virus, Epstein-Barr virus, rubella virus, measles virus, influenza A virus, parainfluenza virus, influenza B virus, cytomegalovirus, respiratory syncytial virus, hepatitis E virus, group B Streptococcus, Klebsiella pneumoniae, Escherichia coli, Neisseria meningitidis, Streptococcus pneumoniae, Staphylococcus aureus, Salmonella, and Haemophilus influenzae, indicating that this kit has good specificity.

[0182] 3. Repeatability analysis

[0183] The Norovirus GI, GII and GIV pseudoviruses identified by digital PCR were mixed and diluted to 1×10 4The RNA concentrations of 1000 copies / mL and 5000 copies / mL were tested 20 times according to the detection method of Example 3. The corresponding Ct values ​​were recorded, and the mean (M) and standard deviation (SD) of the Ct values ​​of the two concentrations were calculated. The coefficient of variation (CV) was calculated according to the formula CV = (SD / M) × 100%. The results of the repeatability analysis are shown in Table 9. As can be seen from the test results, the CV of the Ct values ​​of each sample tested 20 times in the FAM / VIC / CY5 channel was less than 5%, indicating that the test kit of this application has good detection repeatability.

[0184] Table 9: Repeatability test results

[0185]

[0186] 4. Compliance rate analysis

[0187] Fifty clinical stool samples from suspected patients at a hospital were tested using the test kit and method provided by this application, and in parallel using the primers, probes, and detection method described in patent CN110273027A. The test results are shown in Table 10.

[0188] Table 10: Test results of clinical samples

[0189]

[0190] Using the kit of the present application for testing, 8 of the 50 samples were positive for GI type, 17 were positive for GII type, and 1 was positive for GIV type, while norovirus was detected in the remaining 24 samples. Using the detection method described in patent CN110273027A for testing, 6 of the 50 samples were positive for GI type (2 were not detected), 14 were positive for GII type (3 were not detected), and norovirus was detected in the remaining 30 samples. This shows that the kit of the present application can be effectively used for the rapid detection of norovirus, and its sensitivity is better than the method of patent CN110273027A.

[0191] The above-described embodiments only express several implementation methods of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that, for those skilled in the art, several variations and improvements can be made without departing from the concept of the present application, and these all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of the present application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A primer pair for detecting norovirus, characterized in that: The detection primer pair is selected from one or more pairs of the following primer pairs: A primer pair for detecting Norovirus GI type, comprising: an upstream primer having a sequence as shown in SEQ ID NO.1 to SEQ ID NO.4, and a downstream primer having a sequence as shown in SEQ ID NO.5; A detection primer pair for detecting norovirus GII type, comprising: an upstream primer having a sequence as shown in SEQ ID NO.15 to SEQ ID NO.17, and a downstream primer having a sequence as shown in SEQ ID NO.18; and, A detection primer pair for detecting norovirus GIV type includes: an upstream primer with a sequence as shown in SEQ ID NO.30, and a downstream primer with a sequence as shown in SEQ ID NO.

31.

2. A norovirus detection probe, characterized in that: The detection probe is capable of specifically binding to the amplified fragment of the detection primer pair according to claim 1; Optionally, the detection probe comprises: The GI type probe has a sequence as shown in SEQ ID NO.6; GII type probes with sequences as shown in SEQ ID NO.19 to SEQ ID NO.20; The sequence of the GIV type probe is shown in SEQ ID NO.

32.

3. The detection probe according to claim 2, characterized in that: The detection probes are each independently labeled with a fluorescent reporter group and a fluorescent quencher group, and the fluorescent reporter groups labeled with each detection probe are different.

4. A Norovirus detection kit, characterized in that: The norovirus detection kit comprises the detection primer pair according to claim 1 and / or the detection probe according to claims 2 to 3; Optionally, the detection kit further comprises an internal reference primer pair and an internal reference probe for detecting an internal reference gene.

5. The detection kit according to claim 4, characterized in that The internal reference primer pair includes an upstream primer whose sequence is shown in SEQ ID NO.41 and a downstream primer whose sequence is shown in SEQ ID NO.42; the internal reference probe sequence includes an internal reference probe shown in SEQ ID NO.

43.

6. The detection kit according to any one of claims 4 to 5, characterized in that The detection kit also includes one or more of a Norovirus nucleic acid extract, a Norovirus PCR reaction solution, and a Norovirus PCR enzyme solution.

7. The detection kit according to claim 6, characterized in that The norovirus nucleic acid extract comprises a pH value of 8.2-8.4, 80mM-120mM Tris-HCl buffer, 1.0%-1.4% (v / v) NP-40, 1.8-2.2% (v / v) TritonX-100, 2.4%-2.6% (v / v) Tween-80, 80mM-120mM KCl, 28mM-32mM NaOH, 0.8U / μL-1.2U / μL RNase inhibitor and 120mg / mL-130mg / mL nanomagnetic beads.

8. The detection kit according to claim 6, characterized in that The Norovirus PCR reaction solution includes a pH of 8.8 to 9.2, 48 mM to 52 mM Tris buffer, 80 mM to 120 mM potassium chloride, 3.8 mM to 4.2 mM magnesium sulfate, 0.6% to 0.8% (v / v) Triton X-100, 0.4 mg / mL to 0.8 mg / mL bovine serum albumin, 0.08 U / μL to 0.12 U / μL inorganic pyrophosphatase, 4.8% to 5.2% (v / v) DMSO, and 0.18 mM to 0.22 mM dNTPs; and / or The Norovirus PCR enzyme solution includes 0.50U / μL to 0.60U / μL reverse transcriptase, 0.12U / μL to 0.13U / μL TaqDNA polymerase, 0.8U / μL to 1.2U / μL RNasin, and 0.08U / μL to 0.12U / μL Heat-UDG.

9. The detection kit according to any one of claims 4 to 5 and 7 to 8, characterized in that The detection kit further comprises one or both of a negative control and a positive control; Optionally, the negative control includes physiological saline; the positive control includes a pseudovirus containing Norovirus GI type, a pseudovirus containing Norovirus GII type, a pseudovirus containing Norovirus GIV type, and a pseudovirus containing a specific fragment of an internal reference gene.

10. A method for detecting norovirus typing, characterized in that: include: Extracting the norovirus nucleic acid to be tested from the sample, performing a fluorescent quantitative PCR reaction using the norovirus detection kit according to any one of claims 4 to 9 to obtain an amplification product; as well as Collect fluorescence signals and type norovirus.

11. The method for detecting norovirus typing according to claim 10, characterized in that: The method has one or more of the following features: (1) The sample is a human stool sample or a human vomitus sample; (2) The fluorescent quantitative PCR reaction is a one-step multiplex fluorescent quantitative PCR reaction; (3) The amplification program of the PCR reaction includes: 54°C to 56°C, reaction for 1 min to 3 min; 94°C to 96°C, reaction for 0.5 min to 1.5 min; 93°C to 95°C, reaction for 0.8s to 1.2s, 55°C to 65°C, reaction for 12s to 14s, for a total of 5 cycles; 90°C to 94°C, reaction for 0.8s to 1.2s, 55°C to 65°C, reaction for 12s to 14s, collecting fluorescence signals, for a total of 40 cycles.

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