Cross primer isothermal amplification primer set, system and application for detecting norovirus

Abstract

The application discloses a cross primer isothermal amplification primer group, a system and application for detecting norovirus, and belongs to the technical field of medical detection. The application provides the cross primer isothermal amplification primer group for detecting norovirus, which is composed of an inner loop primer IP, a molecular beacon AMP, a reverse cross primer CPR, a forward cross primer CPF, a reverse primer RB and a forward primer FB. The cross primer isothermal amplification primer group for detecting norovirus is used to detect a nucleic acid sample as a template, and isothermal amplification is carried out by using the above-mentioned primers. After reaction, a result can be directly determined by using a detection analyzer. The application has good specificity, high accuracy, high sensitivity, simple and rapid operation, and is suitable for instant detection of norovirus.

Description

Technical Field

[0001] This invention belongs to the field of medical testing technology, specifically relating to a cross-primer isothermal amplification primer set, system, and application for detecting norovirus. Background Technology

[0002] Norovirus (NoV) is a leading cause of sporadic outbreaks of acute gastroenteritis (AGE) worldwide. NoV is primarily transmitted from person to person or via the fecal-oral route through contaminated food and water, and is highly contagious, infecting even at low doses. It is estimated that NoV infects approximately 700 million people and causes about 200,000 deaths globally each year, with over 70,000 of those deaths occurring among children in developing countries. Therefore, NoV infection not only threatens the health and lives of people worldwide, especially children, but also imposes a heavy medical and economic burden.

[0003] Norovirus (Norovirus) belongs to the Caliciviridae family and is a non-enveloped virus with a single-stranded RNA genome of approximately 7.4-7.7 kb in length. It contains three open reading frames (ORFs): ORF1 encodes six non-structural proteins, including an RNA-dependent RNA polymerase (RdRp); ORF2 encodes the major structural protein (VP1); and ORF3 encodes the minor structural protein (VP2). Based on the complete capsid amino acid sequence, Norovirus is classified into 10 genomes (GI-GX), of which GI, GII, and GIV infect humans. GII.4 and GII.3 were the dominant genotypes in norovirus outbreaks and sporadic cases in my country over the past decade, while from 2024-2025 onwards, GII.17 has gradually replaced GII.4 as the dominant genotype. NoV often recombines in the ORF1-ORF2 overlap region, thus a dual nomenclature system exists based on the RdRp region (polymerase / P genotype) and the VP1 region (capsid / G genotype). In nature, strains formed by various combinations of the P and G genotypes can also be found. Therefore, the co-circulation of multiple NoV genotypes, coupled with the lack of suitable in vitro culture systems and mature animal infection models, makes the development of vaccines and antiviral drugs extremely difficult.

[0004] Currently, the most commonly used clinical method for detecting Novovirus (NOV) is polymerase chain reaction (PCR), with real-time quantitative PCR (RT-qPCR) being the most widely used due to its high sensitivity and specificity. However, PCR technology has limitations, such as long processing time, expensive equipment, and high requirements for laboratory environment and operator expertise, making it often difficult to implement in outpatient and emergency department testing. Various nucleic acid-based point-of-care testing (POCT) systems have emerged and been rapidly adopted in outpatient and emergency department testing scenarios. However, there is currently no POCT system specifically for NOV. Therefore, there is an urgent need for a localized, low-cost POCT testing system to provide more timely and convenient services for the diagnosis and treatment of clinical NOV-infected patients. Summary of the Invention

[0005] In view of the above, the purpose of this invention is to provide a cross-primer isothermal amplification primer set, system, and application for detecting norovirus. This invention aims to develop a Norovirus point-of-care testing (POCT) system based on CPA technology, taking into account the epidemiological characteristics of Norovirus infection in children. This invention searches Norovirus gene sequences in gene databases, analyzes pathogen-specific gene targets, designs specific and sensitive detection primers targeting these targets, and sets reasonable internal control targets according to sample conditions, resulting in a product design that achieves optimal detection results for standard strain sequences.

[0006] To achieve the above objectives, the present invention specifically adopts the following technical solution: The first objective of this invention is to provide a cross-primer isothermal amplification primer set for detecting norovirus, the cross-primer isothermal amplification primer set comprising at least one of primer set A, primer set B, and primer set C; Primer set A consists of inner loop primer IP1, molecular beacon AMP1, reverse cross primer CPR1, forward cross primer CPF1, reverse primer RB1, and forward primer FB1, with their nucleotide sequences shown in SEQ ID NO:1~SEQ ID NO:6. Primer set B consists of inner loop primer IP2, molecular beacon AMP2, reverse cross primer CPR2, forward cross primer CPF2, reverse primer RB2, and forward primer FB2, and their nucleotide sequences are shown in SEQ ID NO:7~SEQ ID NO:12 in sequence. Primer set C consists of inner loop primer IP3, molecular beacon AMP3, reverse cross primer CPR3, forward cross primer CPF3, reverse primer RB3, and forward primer FB3, and their nucleotide sequences are shown in SEQ ID NO:13~SEQ ID NO:18.

[0007] Furthermore, the norovirus type is one or more of GII.3, GII.4, or GII.17.

[0008] Furthermore, when detecting GII.3 alone, the cross-primer isothermal amplification primer set includes primer set A; when detecting GII.4 alone, the cross-primer isothermal amplification primer set includes primer set B; and when detecting GII.17 alone, the cross-primer isothermal amplification primer set includes primer set C.

[0009] Furthermore, when simultaneously detecting GII.3 and GII.4, the cross-primer isothermal amplification primer set includes primer set A and primer set B; when simultaneously detecting GII.3 and GII.17, the cross-primer isothermal amplification primer set includes primer set A and primer set C; when simultaneously detecting GII.4 and GII.17, the cross-primer isothermal amplification primer set includes primer set B and primer set C.

[0010] Furthermore, when simultaneously detecting GII.3, GII.4, and GII.17, the cross-primer isothermal amplification primer set includes primer set A, primer set B, and primer set C.

[0011] Furthermore, the 5' end of the molecular beacon is modified with 6-FAM.

[0012] Further, the molar ratio of the forward primer, reverse primer, reverse cross primer, forward cross primer, intraloop primer, and molecular beacon is (0.95-1.05):(0.95-1.05):(8.5-9.5):(8.5-9.5):(6.5-7.5):(0.95-1.05).

[0013] The second objective of this invention is to provide a cross-primer isothermal amplification reagent for detecting norovirus, which consists of the aforementioned primer set, reaction buffer, enzyme, and DEPC water. The final concentrations of the forward primer, reverse primer, and molecular beacon in the amplification reagent are all 0.1 μM. The final concentrations of both the reverse cross primer and the forward cross primer in the amplification reagent were 0.9 μM. The final concentration of the intraloop primers in the amplification reagent was 0.7 μM.

[0014] The third objective of this invention is to provide a cross-primer isothermal amplification system for detecting norovirus, comprising an online nucleic acid extraction and purification section and an online nucleic acid reaction and amplification section; The online nucleic acid extraction and purification section includes a lysis zone, a magnetic bead adsorption zone, and a washing zone. The online nucleic acid reaction amplification section includes a fluorescent probe and a primer amplification region containing the aforementioned primer set or the aforementioned amplification reagent.

[0015] The fourth objective of this invention is to provide a method for using the above-mentioned cross-primer isothermal amplification system, comprising the following steps: The sample to be tested was extracted, purified, and amplified using a cross-primer isothermal amplification system to obtain the amplification product. The amplification product is placed in a nucleic acid amplification detection and analysis instrument, and the fluorescent probe specifically binds to a target sequence in the amplification product to generate a fluorescent signal; If the analyzer detects a fluorescent signal, the sample is infected with norovirus; if the analyzer does not detect a fluorescent signal, the sample is not infected with norovirus.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. The detection principle of this invention is cross-priming amplification (CPA), which operates under isothermal conditions and eliminates the need for the denaturation-annealing-extension thermal cycle of conventional PCR, resulting in a very fast reaction time. Preliminary clinical validation results indicate that positive reactions can occur as early as 5 minutes and no later than 30 minutes, significantly faster than the currently used clinical norovirus quantitative PCR detection system (2-3 hours).

[0017] 2. The detection system of this invention, equipped with a fully automated detection tube, integrates nucleic acid extraction and purification, isothermal amplification, and detection and analysis functions. It is a novel molecular detection system with advantages such as concentrated functions, portability, simple operation, and prevention of cross-contamination.

[0018] 3. After validation with clinical samples, the detection system of this invention was compared with the clinical test kit for norovirus (based on the principle of quantitative real-time PCR). The positive concordance rate was 100%, the negative concordance rate was 97.0%, and the total concordance rate was 99.1%. Moreover, the detection system of this invention can detect low-concentration NoV positive samples that are missed by the clinical test kit (the detection limit is as low as 15.8 copies / μL), so the results are more accurate and reliable.

[0019] 4. Conventional PCR technology is characterized by expensive equipment and high requirements for laboratory environment and operator expertise, making it difficult to implement in outpatient and emergency department testing. However, the detection system of this invention does not require a thermal cycler; users only need to perform a single sample addition operation to complete the nucleic acid detection of norovirus in clinical samples with a "one-click" process. Therefore, the requirements for operator skills and equipment are not high, making this method highly suitable for the turn-around time (TAT) requirements in hospital pathogen testing practices and applicable to point-of-care testing. Attached Figure Description

[0020] Figure 1 This is a diagram of the reaction device for the norovirus nucleic acid detection system of the present invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with embodiments. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0022] 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. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0023] Example 1: Design of cross-primer isothermal amplification primers Eighteen primers and probes were designed for common norovirus serotypes (GII.3, GII.4, GII.17), and the primer and probe sequences are shown in Table 1. Specifically, IP1, AMP1, CPR1, CPF1, RB1, and FB1 are the inner-loop primer, molecular beacon, reverse cross primer, forward cross primer, reverse primer, and forward primer for GII.3, respectively; IP2, AMP2, CPR2, CPF2, RB2, and FB2 are the inner-loop primer, molecular beacon, reverse cross primer, forward cross primer, reverse primer, and forward primer for GII.4, respectively; and IP3, AMP3, CPR3, CPF3, RB3, and FB3 are the inner-loop primer, molecular beacon, reverse cross primer, forward cross primer, reverse primer, and forward primer for GII.17, respectively.

[0024] Table 1 Primer and probe sequences

[0025] Example 2: Application of cross-primer isothermal amplification primers in the detection of norovirus The product is a norovirus nucleic acid detection system based on the principle of cross-primer amplification, consisting of two parts: online nucleic acid extraction and purification, and online nucleic acid reaction amplification. The detection tube is a reaction device integrating nucleic acid extraction and purification and amplification reaction. Figure 1 The reaction amplification system mainly consists of forward primers (FB), reverse primers (RB), cross-bridge primers (CPR), cross-bridge primers (CPF), internal primers (IP), molecular beacons (AMP), Bst enzyme buffer (containing 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8), and Bst enzyme for common norovirus subtypes (GII.3, GII.4, and GII.17). The content of each component is shown in Table 2.

[0026] Table 2 Final concentration / volume of each component in the reaction amplification system

[0027] Detection Procedure: The test tube containing the sample is inserted into the nucleic acid amplification and analysis instrument. The heating block in the lysis zone of the test tube heats up, releasing the nucleic acid from the sample into the solution. After the released nucleic acid is adsorbed by the magnetic beads, it is dragged from the lysis zone by a magnet through the hydrophobic layer to the cleaning zone. Impurities on the magnetic beads are washed away by the cleaning solution. The beads are then dragged from the cleaning zone by the magnet through the hydrophobic layer to the amplification zone, where the nucleic acid is eluted by the elution solution, completing the nucleic acid purification. The purified nucleic acid and reaction reagents are amplified through the temperature-controlled amplification zone. Simultaneously, the fluorescent probe specifically binds to a target sequence in the primer amplification zone, generating a fluorescence signal. The instrument's optical module detects the fluorescence signal, which is then analyzed by the CPU to calculate the fluorescence values ​​of different channels. The system automatically generates the amplification curve, Tt value, and results.

[0028] A total of 152 stool samples were collected. First, conventional PCR followed by first-generation sequencing detected 45 GII.3 positive samples, 39 GII.4 positive samples, 32 GII.17 positive samples, 12 samples that were not GII.3, GII.4, or GII.17 positive, and 24 negative samples. After screening out samples that were not GII.3, GII.4, or GII.17 positive, the remaining 140 stool samples were randomly shuffled and tested with the kit and the system of this invention, along with 8 template-free control samples.

[0029] After identification using the Hubei Lande Norovirus Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method), the results were classified as positive or negative. Then, the results were verified using the system of this invention, and the analysis results were summarized.

[0030] The clinical test kit results were: 115 positive samples, 25 negative samples; and none of the 8 template-free control samples were detected.

[0031] The above samples were grouped and retested using the norovirus nucleic acid detection system of this invention. The results were as follows: In the clinical kit positive group (115 cases), all samples tested positive with this product. In the clinical kit negative group (25 cases), one sample tested positive with this product (confirmed as GII.3 positive after sequencing identification), and the remaining 24 samples were negative.

[0032] As can be seen, the product of this invention achieves a 100% detection rate for common norovirus serotypes GII.3, GII.4, and GII.17; the positive concordance rate between the clinical kit and this product is 100%, the negative concordance rate is 97.0%, and the overall concordance rate is 99.1%. Compared with ordinary PCR technology, the detection system of this invention does not require expensive instruments such as thermal cyclers or professional operators. It only requires one step of sample addition to complete the nucleic acid detection of norovirus in the sample with one click. At the same time, it can also detect low-concentration NoV positive samples that are missed by clinical kits, so the results are more accurate and reliable.

[0033] Comparative Example 1: Design of cross-primers for isothermal amplification Several primers and probes were designed for common norovirus subtypes (GII.3, GII.4, GII.17), and the designed primer and probe sequences are shown in Table 3-5.

[0034] Table 3

[0035] Table 4

[0036] Table 5

[0037] The same actual sample was tested as in Example 2, and the results were as follows: Of the 45 GII.3 positive samples, 34 were positive in this comparative study, with a concordance rate of less than 80%; of the 39 GII.4 positive samples and 32 GII.17 positive samples, none were detected in this comparative study.

[0038] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they are not intended to limit the present invention. It should be noted that various changes and modifications can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the concept and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A cross-primer isothermal amplification primer set for detecting norovirus, characterized in that, The cross-primer isothermal amplification primer set includes at least one of primer set A, primer set B, and primer set C; Primer set A consists of inner loop primer IP1, molecular beacon AMP1, reverse cross primer CPR1, forward cross primer CPF1, reverse primer RB1, and forward primer FB1, with their nucleotide sequences shown in SEQ ID NO:1~SEQ ID NO:

6. Primer set B consists of inner loop primer IP2, molecular beacon AMP2, reverse cross primer CPR2, forward cross primer CPF2, reverse primer RB2, and forward primer FB2, and their nucleotide sequences are shown in SEQ ID NO:7~SEQ ID NO:12 in sequence. Primer set C consists of inner loop primer IP3, molecular beacon AMP3, reverse cross primer CPR3, forward cross primer CPF3, reverse primer RB3, and forward primer FB3, and their nucleotide sequences are shown in SEQ ID NO:13~SEQ ID NO:

18.

2. The cross-primer isothermal amplification primer set for detecting norovirus according to claim 1, characterized in that, The norovirus type is one or more of GII.3, GII.4, or GII.

17.

3. The cross-primer isothermal amplification primer set for detecting norovirus according to claim 1, characterized in that, When detecting GII.3 alone, the cross-primer isothermal amplification primer set includes primer set A; when detecting GII.4 alone, the cross-primer isothermal amplification primer set includes primer set B; when detecting GII.17 alone, the cross-primer isothermal amplification primer set includes primer set C.

4. The cross-primer isothermal amplification primer set for detecting norovirus according to claim 1, characterized in that, When simultaneously detecting GII.3 and GII.4, the cross-primer isothermal amplification primer set includes primer set A and primer set B; when simultaneously detecting GII.3 and GII.17, the cross-primer isothermal amplification primer set includes primer set A and primer set C; when simultaneously detecting GII.4 and GII.17, the cross-primer isothermal amplification primer set includes primer set B and primer set C.

5. The cross-primer isothermal amplification primer set for detecting norovirus according to claim 1, characterized in that, When simultaneously detecting GII.3, GII.4, and GII.17, the cross-primer isothermal amplification primer set includes primer set A, primer set B, and primer set C.

6. The cross-primer isothermal amplification primer set for detecting norovirus according to claim 1, characterized in that, The 5' end of each molecular beacon is modified with 6-FAM.

7. The cross-primer isothermal amplification primer set for detecting norovirus according to claim 1, characterized in that, The molar ratio of the forward primer, reverse primer, reverse cross primer, forward cross primer, intraloop primer, and molecular beacon is (0.95-1.05):(0.95-1.05):(8.5-9.5):(8.5-9.5):(6.5-7.5):(0.95-1.05).

8. A cross-primer isothermal amplification reagent for detecting norovirus, comprising the primer set, reaction buffer, enzyme, and DEPC water as described in any one of claims 1-7; The final concentrations of the forward primer, reverse primer, and molecular beacon in the amplification reagent are all 0.1 μM. The final concentrations of both the reverse cross primer and the forward cross primer in the amplification reagent were 0.9 μM. The final concentration of the intraloop primers in the amplification reagent was 0.7 μM.

9. A cross-primer isothermal amplification system for detecting norovirus, including an online nucleic acid extraction and purification section and an online nucleic acid reaction and amplification section; The online nucleic acid extraction and purification section includes a lysis zone, a magnetic bead adsorption zone, and a washing zone. The online nucleic acid reaction amplification portion includes a fluorescent probe and a primer amplification region containing any one of the primer sets according to claims 1-7 or the amplification reagent according to claim 8.

10. A method of using the cross-primer isothermal amplification system of claim 9, comprising the following steps: The sample to be tested was extracted, purified, and amplified using a cross-primer isothermal amplification system to obtain the amplification product. The amplification product is placed in a nucleic acid amplification detection and analysis instrument, and the fluorescent probe specifically binds to a target sequence in the amplification product to generate a fluorescent signal; If the analyzer detects a fluorescent signal, the sample is infected with norovirus; if the analyzer does not detect a fluorescent signal, the sample is not infected with norovirus.

Images