Primer pairs, kits and methods for detecting rhinovirus

Abstract

The present disclosure provides a primer pair for detecting rhinovirus, comprising a forward primer and a reverse primer directed to a 5' untranslated region (5' UTR), and the 5' UTR comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 21 to 25. The forward primer has a length of 12 to 30 nucleotides and is corresponding to a nucleotide sequence between positions 1 to 70 of any one of SEQ ID NOs: 21 to 25. The reverse primer has a length of 12 to 30 nucleotides and is complementary to a nucleotide sequence between positions 180 to 204 of any one of SEQ ID NOs: 21 to 25. The present disclosure also provides a kit for detecting rhinovirus comprising the aforementioned primer pair, and a method for detecting rhinovirus using the aforementioned primer pair.

Description

[Technical Field]

[0001] This disclosure relates to a primer pair, kit, and method for detecting rhinovirus. [Previous Technology]

[0002] Rhinovirus (RNV) is a common respiratory pathogen and the most common cause of the common cold. It is named for its tendency to infect the nose and cause inflammation. Rhinovirus does not have a specific seasonal pattern and can cause infection year-round, but it is more prevalent in early autumn and spring. Common symptoms of rhinovirus include fever, sore throat, runny nose, cough, and body aches. Infections are usually mild, but in patients with asthma, otitis media, bronchitis, or pneumonia, infection can exacerbate their condition and lead to serious illness.

[0003] Rhinoviruses can be classified into types A, B, and C based on their serotypes, with type A having the highest detection rate. Clinical studies have observed a high probability of co-infection with other viruses, bacteria, or fungi, and co-infection is strongly associated with severe illness. Reverse transcription polymerase chain reaction (RT-PCR) can be used in urgent diagnostic tests as a reference for clinicians to rapidly diagnose rhinovirus infection.

[0004] Because rhinovirus sequences are highly variable, there are currently no vaccines, targeted drugs, or treatments that can cure rhinovirus-related diseases; therefore, supportive therapy is the most common approach. Testing for rhinovirus infection helps healthcare professionals determine the type of infection in high-risk groups for respiratory failure (such as asthma or pneumonia patients), and is crucial for early diagnosis, treatment, and controlling infection risk.

[0005] In view of this, developing detection tools and methods that can quickly and accurately detect whether someone is infected with rhinovirus is crucial for improving the cure rate of diseases, and it is one of the topics that the industry is currently working hard to research. [Summary of the Invention]

[0006] According to some embodiments of this disclosure, a primer pair for detecting rhinovirus is provided, comprising a forward primer and a reverse primer targeting the 5' untranslated region (5'UTR), wherein the 5'UTR contains a nucleotide sequence as shown in any of SEQ ID NOs:21 to 25. The forward primer is 12 to 30 nucleotides in length and is a nucleotide sequence corresponding to sites 1 to 70 shown in any of SEQ ID NOs:21 to 25. The reverse primer is 12 to 30 nucleotides in length and is a nucleotide sequence complementary to sites 180 to 204 shown in any of SEQ ID NOs:21 to 25.

[0007] According to some embodiments of this disclosure, a kit for detecting rhinovirus is also provided, comprising a forward primer, a reverse primer, and a probe. The forward primer, reverse primer, and probe target the 5' untranslated region (5'UTR), and the 5'UTR contains a nucleotide sequence as shown in any of SEQ ID NOS:21 to 25. The forward primer is 12 to 30 nucleotides in length and corresponds to the nucleotide sequence between sites 1 to 70 shown in any of SEQ ID NOS:21 to 25. The reverse primer is 12 to 30 nucleotides in length and is complementary to the nucleotide sequence between sites 180 to 204 shown in any of SEQ ID NOS:21 to 25.

[0008] According to some embodiments of this disclosure, a method for detecting rhinovirus is further provided, comprising providing a sample and providing a primer pair, the primer pair comprising a forward primer and a reverse primer targeting the 5' untranslated region (5'UTR), and the 5'UTR comprising a nucleotide sequence as shown in any one of SEQ ID NOs:21 to 25. The forward primer is 12 to 30 nucleotides in length and is a nucleotide sequence corresponding to sites 1 to 70 shown in any one of SEQ ID NOs:21 to 25. The reverse primer is 12 to 30 nucleotides in length and is a nucleotide sequence complementary to sites 180 to 204 shown in any one of SEQ ID NOs:21 to 25. The method for detecting rhinovirus further comprises performing a polymerase chain reaction with the sample using the primer pair to obtain a product, and analyzing the product to detect the presence of rhinovirus.

[0009] To make the features or advantages of this disclosure more apparent and understandable, some embodiments are described below in detail with reference to the accompanying drawings. [Attached Image Description]

[0010] Figure 1A This disclosure shows partial fragments (bases 352 to 555) of the 5' UTR sequence of rhinovirus (GenBank: MN749151.1) according to some embodiments of the present disclosure, as well as the designed positions of the forward primer, probe, and reverse primer; Figure 1B This disclosure shows partial fragments (bases 350 to 553) of the 5' UTR sequence of rhinovirus (GenBank: MH517019.1) according to some embodiments of the present disclosure, as well as the designed positions of the forward primer, probe, and reverse primer; Figure 1CThis disclosure shows partial fragments (bases 334 to 538) of the 5' UTR sequence of rhinovirus (GenBank: KY369891.1) according to some embodiments of the present disclosure, as well as the designed positions of the forward primer, probe, and reverse primer; Figure 1D This disclosure shows partial fragments (bases 322 to 524) of the 5' UTR sequence of rhinovirus (GenBank: MZ153247.1) according to some embodiments of the present disclosure, as well as the designed positions of the forward primer, probe, and reverse primer; Figure 1E This disclosure shows partial fragments (bases 368 to 571) of the 5' UTR sequence of rhinovirus (GenBank: EF173424.1) according to some embodiments of the present disclosure, as well as the designed positions of the forward primer, probe, and reverse primer; Figure 2 This displays a temperature curve of a real-time quantitative reverse transcription polymerase chain reaction according to some embodiments of the present disclosure; Figure 3A The display shows amplification curves of real-time quantitative reverse transcription polymerase chain reaction performed using rhinovirus A / C type RNA as a template, according to some embodiments of this disclosure. Figure 3B The display shows amplification curves of real-time quantitative reverse transcription polymerase chain reaction performed using rhinovirus A / C type RNA as a template, according to some embodiments of this disclosure. Figure 3C The display shows amplification curves of real-time quantitative reverse transcription polymerase chain reaction performed using rhinovirus A / C type RNA as a template, according to some embodiments of this disclosure. Figure 3D The display shows amplification curves of real-time quantitative reverse transcription polymerase chain reaction performed using rhinovirus B1 RNA as a template, according to some embodiments of this disclosure. Figure 3E The display shows amplification curves of real-time quantitative reverse transcription polymerase chain reaction performed using rhinovirus B2 RNA as a template, according to some embodiments of this disclosure. Figure 4 This image shows the electrophoresis results of real-time quantitative reverse transcription polymerase chain reaction products according to some embodiments of this disclosure.

Implementation Method

[0011] The primer pairs, kits, and methods for detecting rhinovirus according to embodiments of this disclosure are described in detail below. It should be understood that the following description provides many different embodiments for implementing different aspects of some embodiments of this disclosure, but this is not the only form of implementing or utilizing the specific embodiments of this invention. The embodiments disclosed herein can be combined or substituted with each other where advantageously possible, and other embodiments can be added to one embodiment without further description or explanation. In the following description, many specific details will be set forth in detail to enable the reader to fully understand the embodiments described below. However, embodiments of the invention disclosed herein can also be practiced without such specific details.

[0012] In this document, unless otherwise specified in the text, “a” and “the” may refer to one or more. It should be understood that the terms “comprising,” “including,” “having,” and similar terms as used herein specify the features, regions, integers, steps, operations, components, and / or components described herein, but do not exclude other features, regions, integers, steps, operations, components, and / or components.

[0013] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. It is understood that such terms, for example, as defined in a commonly used dictionary, should be interpreted as having a meaning consistent with the relevant art and the background or context of this disclosure, and should not be interpreted in an idealized or overly formal manner, unless specifically defined in the embodiments of this disclosure.

[0014] According to embodiments of this disclosure, a primer pair, a kit, and a method for detecting rhinovirus are provided. The primer pair targets specific gene fragments of rhinovirus, and performs highly sensitive, highly specific, and rapid detection using these specific gene fragments as amplification targets. According to embodiments of this disclosure, the kit for detecting rhinovirus can withstand interference from high concentrations of human genomic DNA, maintain the detection sensitivity of polymerase chain reaction, and maintain a specific gene fragment sequence coverage of over 75% for the three different rhinovirus types A, B, and C.

[0015] According to some embodiments of this disclosure, a primer pair for detecting rhinovirus is provided, comprising a forward primer and a reverse primer targeting the 5' untranslated region (5'UTR), wherein the 5'UTR contains a nucleotide sequence as shown in any of SEQ ID NOs:21 to 25. The forward primer is 12 to 30 nucleotides in length and is a nucleotide sequence corresponding to sites 1 to 70 shown in any of SEQ ID NOs:21 to 25. The reverse primer is 12 to 30 nucleotides in length and is a nucleotide sequence complementary to sites 180 to 204 shown in any of SEQ ID NOs:21 to 25.

[0016] The forward and reverse primers are designed for at least one of the nucleotide sequences shown in SEQ ID NO:21, 22, 23, 24, and 25. According to some embodiments, a portion of the designed forward primer may correspond simultaneously to one or more of the nucleotide sequences in SEQ ID NO:21, 22, 23, 24, and 25; for example, it may correspond simultaneously to two, three, four, or five of the nucleotide sequences. According to some embodiments, a portion of the designed reverse primer may be complementary to one or more of the nucleotide sequences in SEQ ID NO:21, 22, 23, 24, and 25; for example, it may correspond simultaneously to two, three, four, or five of the nucleotide sequences.

[0017] It should be understood that describing a primer as "corresponding to a nucleotide sequence between specific sites" means that the sequence of the primer is substantially the same as the nucleotide sequence at the specific site, for example, the sequences are completely identical or have at least 85% identity; describing a primer as "complementary to a nucleotide sequence between specific sites" means that the sequence of the primer is substantially complementary to the nucleotide sequence at the specific site, for example, the sequences are completely complementary or have at least 85% sequence complementarity.

[0018] Specifically, according to some implementations, the 5'UTR contains a fragment corresponding to base pairs 352 to 555 of GenBank:MN749151.1 (SEQ ID NO:21). This fragment (SEQ ID NO:21) from base pairs 352 to 555 of GenBank:MN749151.1 can be considered a template for rhinovirus types A and C. Please refer to... Figure 1A , Figure 1A This shows partial fragments of the 5' UTR sequence (GenBank: MN749151.1) and the designed positions of the forward primer, probe, and reverse primer according to some embodiments of this disclosure. Figure 1AAs shown, the forward primer corresponds to the nucleotide sequence between sites 1 to 68 of SEQ ID NO:21, and the reverse primer is complementary to the nucleotide sequence between sites 181 to 204 of SEQ ID NO:21. In other words, the forward primer can be designed from the positions between sites 1 to 68 of SEQ ID NO:21, and the reverse primer can be designed from the positions between sites 181 to 204 of SEQ ID NO:21.

[0019] According to some implementations, the 5'UTR contains a fragment corresponding to base pairs 350 to 553 of GenBank:MH517019.1 (SEQ ID NO:22). This fragment (SEQ ID NO:22) from base pairs 350 to 553 of GenBank:MH517019.1 can be considered another template for rhinovirus types A and C. Please refer to... Figure 1B , Figure 1B This shows partial fragments of the 5' UTR sequence (GenBank: MH517019.1) and the designed positions of the forward primer, probe, and reverse primer according to some embodiments of this disclosure. Figure 1B As shown, the forward primer corresponds to the nucleotide sequence between positions 1 to 70 of SEQ ID NO:22, and the reverse primer is complementary to the nucleotide sequence between positions 180 and 203 of SEQ ID NO:22. In other words, the forward primer can be designed from the positions between positions 1 to 70 of SEQ ID NO:22, and the reverse primer can be designed from the positions between positions 180 and 203 of SEQ ID NO:22.

[0020] According to some implementation schemes, the 5'UTR contains a fragment corresponding to base pairs 334 to 538 of GenBank:KY369891.1 (SEQ ID NO:23). This fragment (SEQ ID NO:23) from base pairs 334 to 538 of GenBank:KY369891.1 can be considered another template for rhinovirus types A and C. Please refer to... Figure 1C , Figure 1C This shows partial fragments of the 5' UTR sequence (GenBank: KY369891.1) and the designed positions of the forward primer, probe, and reverse primer according to some embodiments of this disclosure. Figure 1CAs shown, the forward primer corresponds to the nucleotide sequence between sites 1 and 69 of SEQ ID NO:23, and the reverse primer is complementary to the nucleotide sequence between sites 181 and 204 of SEQ ID NO:23. In other words, the forward primer can be designed from the positions between sites 1 and 69 of SEQ ID NO:23, and the reverse primer can be designed from the positions between sites 181 and 204 of SEQ ID NO:23.

[0021] According to some implementation schemes, the 5'UTR contains a fragment corresponding to base pairs 322 to 524 of GenBank:MZ153247.1 (SEQ ID NO:24). This fragment (SEQ ID NO:24) from base pairs 322 to 524 of GenBank:MZ153247.1 can be considered a template for rhinovirus type B. Please refer to... Figure 1D , Figure 1D This shows partial fragments of the 5' UTR sequence (GenBank: MZ 153247.1) and the designed locations of the forward primer, probe, and reverse primer according to some embodiments of this disclosure. Figure 1D As shown, the forward primer corresponds to the nucleotide sequence between positions 2 and 69 of SEQ ID NO:24, and the reverse primer is complementary to the nucleotide sequence between positions 180 and 203 of SEQ ID NO:24. In other words, the forward primer can be designed from the positions between positions 2 and 69 of SEQ ID NO:24, and the reverse primer can be designed from the positions between positions 180 and 203 of SEQ ID NO:24.

[0022] According to some implementations, the 5'UTR contains a fragment (SEQ ID NO:25) corresponding to base pairs 368 to 571 of GenBank:EF173424.1. This fragment (SEQ ID NO:25) from base pairs 368 to 571 of GenBank:EF173424.1 can be considered another template for rhinovirus type B. Please refer to... Figure 1E , Figure 1E This shows partial fragments of the 5' UTR sequence (GenBank: EF173424.1) and the designed positions of the forward primer, probe, and reverse primer according to some embodiments of this disclosure. Figure 1EAs shown, the forward primer corresponds to the nucleotide sequence between positions 46 and 69 of SEQ ID NO:25, and the reverse primer is complementary to the nucleotide sequence between positions 180 and 203 of SEQ ID NO:25. In other words, the forward primer can be designed from the positions between positions 46 and 69 of SEQ ID NO:25, and the reverse primer can be designed from the positions between positions 180 and 203 of SEQ ID NO:25.

[0023] As mentioned above, the length of the forward primer can be 12 to 30 nucleotides, and the length of the reverse primer can be 12 to 30 nucleotides. According to some embodiments, the length of the forward primer can be 14 to 22 nucleotides, for example, 15, 16, 17, 18, 19, 20, or 21 nucleotides. According to some embodiments, the length of the reverse primer can be 16 to 23 nucleotides, for example, 17, 18, 19, 20, 21, or 22 nucleotides.

[0024] According to some embodiments, the forward primer may comprise a nucleotide sequence as shown in any one of SEQ ID NOs:1 to 13. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:1. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:2. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:3. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:4. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:5. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:6. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:7. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:8. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:9. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:10. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:11. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:12. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:13.

[0025] According to some embodiments, the nucleotide sequences shown in SEQ ID NOs:1-13 may also allow for some degree of variation. For example, according to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:1, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:1, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:2, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:2, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:3, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:3, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:4, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:4, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:5, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:5, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:6, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:6, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:7, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:7, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:8, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:8, but is not limited thereto.According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:9, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:9, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:10, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:10, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:11, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:11, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:12, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:12, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:13, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:13, but is not limited thereto.

[0026] In addition, according to some implementation schemes, the nucleotide sequence of the forward primer may contain modifications of nucleic acid analogs, such as locked nucleic acid (LNA) modifications, which can increase the melting temperature (Tm value) of the primer.

[0027] According to some embodiments, the reverse primer may comprise a nucleotide sequence as shown in any of SEQ ID NOs:14-16. According to some embodiments, the reverse primer consists of the nucleotide sequence shown in SEQ ID NO:14. According to some embodiments, the reverse primer consists of the nucleotide sequence shown in SEQ ID NO:15. According to some embodiments, the reverse primer consists of the nucleotide sequence shown in SEQ ID NO:16.

[0028] According to some embodiments, the nucleotide sequences shown in SEQ ID NOs:14-16 may also allow for some degree of variation. For example, according to some embodiments, the reverse primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:14, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:14, but is not limited thereto. According to some embodiments, the reverse primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:15, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:15, but is not limited thereto. According to some embodiments, the reverse primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:16, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:16, but is not limited thereto.

[0029] Furthermore, according to some embodiments, the forward and reverse primers are used for polymerase chain reaction (PCR). Polymerase chain reaction is a molecular biology technique that utilizes primer pairs with oligonucleotide sequences to amplify specific deoxyribonucleic acid (DNA) fragments. It should be understood that the primer pairs provided in this disclosure can be applied to various polymerase chain reaction-based techniques. According to some embodiments, the polymerase chain reaction may include, but is not limited to, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR).

[0030] According to some embodiments of this disclosure, a kit for detecting rhinovirus is provided, comprising a forward primer, a reverse primer, and a probe, wherein the forward primer, reverse primer, and probe target the 5' untranslated region (5'UTR), and the 5'UTR contains a nucleotide sequence as shown in any one of SEQ ID NOs:21 to 25. The forward primer is 12 to 30 nucleotides in length and corresponds to the nucleotide sequence between sites 1 to 70 shown in any one of SEQ ID NOs:21 to 25. The reverse primer is 12 to 30 nucleotides in length and is complementary to the nucleotide sequence between sites 180 to 204 shown in any one of SEQ ID NOs:21 to 25. The probe is 13 to 21 nucleotides in length and corresponds to the nucleotide sequence between sites 83 to 104 shown in any one of SEQ ID NOs:21 to 25.

[0031] In detail, such as Figure 1A As shown, according to some embodiments, the forward primer may correspond to the nucleotide sequence between sites 1 to 68 of SEQ ID NO:21, the reverse primer may be complementary to the nucleotide sequence between sites 181 to 204 of SEQ ID NO:21, and the probe may correspond to the nucleotide sequence between sites 83 to 103 of SEQ ID NO:21. Figure 1B As shown, according to some embodiments, the forward primer may correspond to the nucleotide sequence between sites 1 to 70 of SEQ ID NO:22, the reverse primer may be complementary to the nucleotide sequence between sites 180 and 203 of SEQ ID NO:22, and the probe may correspond to the nucleotide sequence between sites 84 and 104 of SEQ ID NO:22. Figure 1C As shown, according to some embodiments, the forward primer may correspond to the nucleotide sequence between sites 1 to 69 of SEQ ID NO:23, the reverse primer may be complementary to the nucleotide sequence between sites 181 to 204 of SEQ ID NO:23, and the probe may correspond to the nucleotide sequence between sites 84 to 104 of SEQ ID NO:23. Figure 1D As shown, the forward primer corresponds to the nucleotide sequence between sites 2 and 69 of SEQ ID NO:24, the reverse primer is complementary to the nucleotide sequence between sites 180 and 203 of SEQ ID NO:24, and the probe corresponds to the nucleotide sequence between sites 83 and 103 of SEQ ID NO:24. Figure 1EAs shown, the forward primer corresponds to the nucleotide sequence between sites 46 and 69 of SEQ ID NO:25, the reverse primer is complementary to the nucleotide sequence between sites 180 and 203 of SEQ ID NO:25, and the probe corresponds to the nucleotide sequence between sites 83 and 103 of SEQ ID NO:25.

[0032] Furthermore, as mentioned above, the length of the forward primer can be 12 to 30 nucleotides, and the length of the reverse primer can be 12 to 30 nucleotides. According to some embodiments, the length of the forward primer can be 14 to 22 nucleotides, for example, 15, 16, 17, 18, 19, 20, or 21 nucleotides. According to some embodiments, the length of the reverse primer can be 16 to 23 nucleotides, for example, 17, 18, 19, 20, 21, or 22 nucleotides. According to some embodiments, the length of the probe can be 13 to 21 nucleotides, for example, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0033] According to some embodiments, the forward primer may comprise the nucleotide sequence shown in any one of SEQ ID NOs:1-13. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:1. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:2. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:3. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:4. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:5. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:6. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:7. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:8. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:9. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:10. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:11. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:12. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:13.

[0034] According to some embodiments, the nucleotide sequences shown in SEQ ID NOs:1-13 may also allow for some degree of variation. For example, according to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:1, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:1, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:2, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:2, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:3, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:3, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:4, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:4, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:5, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:5, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:6, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:6, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:7, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:7, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:8, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:8, but is not limited thereto.According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:9, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:9, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:10, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:10, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:11, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:11, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:12, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:12, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:13, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:13, but is not limited thereto.

[0035] In addition, according to some implementation schemes, the nucleotide sequence of the forward primer may contain modifications of nucleic acid analogs, such as locked nucleic acid (LNA) modifications, which can increase the melting temperature (Tm value) of the primer.

[0036] According to some embodiments, the reverse primer may comprise a nucleotide sequence as shown in any of SEQ ID NOs:14-16. According to some embodiments, the reverse primer consists of the nucleotide sequence shown in SEQ ID NO:14. According to some embodiments, the reverse primer consists of the nucleotide sequence shown in SEQ ID NO:15. According to some embodiments, the reverse primer consists of the nucleotide sequence shown in SEQ ID NO:16.

[0037] According to some embodiments, the nucleotide sequences shown in SEQ ID NOs:14-16 may also allow for some degree of variation. For example, according to some embodiments, the reverse primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:14, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:14, but is not limited thereto. According to some embodiments, the reverse primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:15, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:15, but is not limited thereto. According to some embodiments, the reverse primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:16, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:16, but is not limited thereto.

[0038] According to some embodiments, the probe may comprise a nucleotide sequence as shown in SEQ ID NO:17. According to some embodiments, the probe consists of a nucleotide sequence as shown in SEQ ID NO:17. According to some embodiments, the nucleotide sequence shown in SEQ ID NO:17 may also allow for some degree of variation. For example, according to some embodiments, the probe may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:17, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:17, but is not limited thereto.

[0039] Furthermore, according to some embodiments, forward primers, reverse primers, and probes are used for polymerase chaining reactions. According to some embodiments, the polymerase chaining reaction may include, but is not limited to, real-time quantitative polymerase chaining reactions, such as real-time quantitative reverse transcription polymerase chaining reactions. According to some embodiments, the real-time polymerase chaining reaction used is a probe-based fluorescent system; therefore, before using the primer pair to perform polymerase chaining reactions with the sample to obtain the product, a further hybridization reaction is performed on the sample using the probe, causing the probe to bind to the target sequence. That is, the primer pair, probe, and sample are subjected to polymerase chaining reactions together to obtain the product.

[0040] Specifically, according to some embodiments, the 5' end of the probe can be linked to a reporter dye, and the 3' end of the probe can be linked to a quencher. During the PCR amplification reaction, the probe is cleaved, causing the reporter dye and quencher to separate, thus allowing the fluorescence emitted by the reporter dye to be detected. According to some embodiments, the reporter dye may contain Fluorescein (FAM), HEX, Texas Red, ROX, Cy5, or other suitable fluorescent groups, and the quencher may contain BHQ, TAMRA, DABCYL, or other suitable groups, but this disclosure is not limited thereto.

[0041] In addition, according to some embodiments, the rhinovirus detection kit also includes forward and reverse primers targeting the nucleic acid sequence of the human gene, wherein the forward primer contains the nucleotide sequence shown in SEQ ID NO:18 and the reverse primer contains the nucleotide sequence shown in SEQ ID NO:19. According to some embodiments, the rhinovirus detection kit also includes a probe targeting the nucleic acid sequence of the human gene, used in conjunction with the aforementioned forward and reverse primers targeting the nucleic acid sequence of the human gene, and the probe contains the nucleotide sequence shown in SEQ ID NO:20. The forward and reverse primers targeting the nucleic acid sequence of the human gene can serve as an internal control to confirm correct sample collection. According to some embodiments, the human gene includes the RNase P gene, which is a housekeeping gene in the human body. The nucleic acid sequence of the human gene may contain a nucleotide sequence encoding ribonuclease P (RNase P).

[0042] According to some implementation schemes, the forward primers, reverse primers, and probes of the kit for detecting rhinovirus can be in freeze-dried form and reconstituted in water for use when needed.

[0043] According to some embodiments of this disclosure, a method for detecting rhinovirus is provided, comprising the steps of providing a sample and providing a primer pair. Specifically, the primer pair comprises a forward primer and a reverse primer targeting the 5' untranslated region (5'UTR), and the 5'UTR comprises a nucleotide sequence as shown in any one of SEQ ID NOs:21 to 25. The forward primer is 12 to 30 nucleotides in length and corresponds to the nucleotide sequence between sites 1 to 70 shown in any one of SEQ ID NOs:21 to 25. The reverse primer is 12 to 30 nucleotides in length and is complementary to the nucleotide sequence between sites 180 to 204 shown in any one of SEQ ID NOs:21 to 25.

[0044] In detail, such as Figure 1A As shown, according to some embodiments, the forward primer may correspond to the nucleotide sequence between sites 1 to 68 of SEQ ID NO:21, and the reverse primer may be complementary to the nucleotide sequence between sites 181 to 204 of SEQ ID NO:21. Figure 1B As shown, according to some embodiments, the forward primer may correspond to the nucleotide sequence between sites 1 to 70 of SEQ ID NO:22, and the reverse primer may be complementary to the nucleotide sequence between sites 180 to 203 of SEQ ID NO:22. Figure 1C As shown, according to some embodiments, the forward primer may correspond to the nucleotide sequence between sites 1 to 69 of SEQ ID NO:23, and the reverse primer may be complementary to the nucleotide sequence between sites 181 to 204 of SEQ ID NO:23. Figure 1D As shown, the forward primer corresponds to the nucleotide sequence between sites 2 and 69 of SEQ ID NO:24, and the reverse primer is complementary to the nucleotide sequence between sites 180 and 203 of SEQ ID NO:24. Figure 1E As shown, the forward primer corresponds to the nucleotide sequence between sites 46 and 69 of SEQ ID NO:25, and the reverse primer is complementary to the nucleotide sequence between sites 180 and 203 of SEQ ID NO:25.

[0045] Furthermore, as mentioned above, the length of the forward primer can be 12 to 30 nucleotides, and the length of the reverse primer can be 12 to 30 nucleotides. According to some embodiments, the length of the forward primer can be 14 to 22 nucleotides, for example, 15, 16, 17, 18, 19, 20, or 21 nucleotides. According to some embodiments, the length of the reverse primer can be 16 to 23 nucleotides, for example, 17, 18, 19, 20, 21, or 22 nucleotides.

[0046] According to some embodiments, the forward primer may comprise the nucleotide sequence shown in any one of SEQ ID NOs:1-13. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:1. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:2. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:3. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:4. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:5. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:6. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:7. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:8. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:9. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:10. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:11. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:12. According to some embodiments, the forward primer consists of the nucleotide sequence shown in SEQ ID NO:13.

[0047] According to some embodiments, the nucleotide sequences shown in SEQ ID NOs:1-13 may also allow for some degree of variation. For example, according to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:1, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:1, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:2, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:2, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:3, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:3, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:4, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:4, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:5, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:5, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:6, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:6, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:7, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:7, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:8, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:8, but is not limited thereto.According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:9, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:9, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:10, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:10, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:11, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:11, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:12, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:12, but is not limited thereto. According to some embodiments, the forward primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:13, for example, it may consist of a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:13, but is not limited thereto.

[0048] In addition, according to some implementation schemes, the nucleotide sequence of the forward primer may contain modifications of nucleic acid analogs, such as locked nucleic acid (LNA) modifications, which can increase the melting temperature (Tm value) of the primer.

[0049] According to some embodiments, the reverse primer may comprise a nucleotide sequence as shown in any of SEQ ID NOs:14-16. According to some embodiments, the reverse primer consists of the nucleotide sequence shown in SEQ ID NO:14. According to some embodiments, the reverse primer consists of the nucleotide sequence shown in SEQ ID NO:15. According to some embodiments, the reverse primer consists of the nucleotide sequence shown in SEQ ID NO:16.

[0050] According to some embodiments, the nucleotide sequences shown in SEQ ID NOs:14-16 may also allow for some degree of variation. For example, according to some embodiments, the reverse primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:14, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:14, but is not limited thereto. According to some embodiments, the reverse primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:15, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:15, but is not limited thereto. According to some embodiments, the reverse primer may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:16, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:16, but is not limited thereto.

[0051] The sample may include samples from a variety of different sources. According to some implementation schemes, the source of the sample may include saliva samples, sputum samples, nasal swab samples, throat swab samples, nasopharyngeal samples, body fluid samples, other suitable samples, or combinations thereof.

[0052] Furthermore, the method for detecting rhinovirus also includes the following steps: performing a polymerase chain reaction with a sample using a primer pair to obtain a product, and analyzing the product to detect the presence of rhinovirus. According to some embodiments, the step of performing a polymerase chain reaction with a sample using a primer pair to obtain a product includes performing a polymerase chain reaction such that the primer pair amplifies the nucleotide sequence of the 5'UTR in the rhinovirus to obtain the product. According to some embodiments, the polymerase chain reaction may include, but is not limited to, a real-time quantitative reverse transcription polymerase chain reaction.

[0053] According to some embodiments, the method for detecting rhinovirus further includes providing a probe. According to some embodiments, the probe is 13 to 21 nucleotides in length and is a nucleotide sequence corresponding to the 83rd to 104th sites shown in any of SEQ ID NOs:21 to 25. According to some embodiments, the probe may comprise a nucleotide sequence as shown in SEQ ID NO:17. According to some embodiments, the probe consists of the nucleotide sequence shown in SEQ ID NO:17. According to some embodiments, the nucleotide sequence shown in SEQ ID NO:17 may also allow for some degree of variation. For example, according to some embodiments, the probe may consist of a nucleotide sequence having at least 85% identity with SEQ ID NO:17, such as a nucleotide sequence having at least 88%, 90%, 92%, 95%, 98%, or 99% identity with SEQ ID NO:17, but is not limited thereto.

[0054] According to some embodiments, the 5' end of the probe can be linked to a reporter dye, and the 3' end of the probe can be linked to a quencher. During the PCR amplification reaction, the probe is cleaved, causing the reporter dye to separate from the quencher, thus allowing the fluorescence emitted by the reporter dye to be detected. According to some embodiments, the reporter dye may contain Fluorescein (FAM), HEX, Texas Red, ROX, Cy5, or other suitable fluorescent groups, and the quencher may contain BHQ, TAMRA, DABCYL, or other suitable groups, but this disclosure is not limited thereto.

[0055] Furthermore, according to some embodiments, the method for detecting rhinovirus also includes providing forward and reverse primers for a nucleic acid sequence targeting a human gene, wherein the forward primer contains the nucleotide sequence shown in SEQ ID NO:18, and the reverse primer contains the nucleotide sequence shown in SEQ ID NO:19. According to some embodiments, the method for detecting rhinovirus also includes providing a probe for a nucleic acid sequence targeting a human gene, used in conjunction with the aforementioned forward and reverse primers for a nucleic acid sequence targeting a human gene, wherein the probe contains the nucleotide sequence shown in SEQ ID NO:20. The forward and reverse primers and the probe for the nucleic acid sequence targeting a human gene can serve as an internal control group to confirm correct sample collection. According to some embodiments, the human gene includes an RNase P gene, and the nucleic acid sequence of the human gene may contain a nucleotide sequence encoding RNase P.

[0056] To make the above and other objects, features and advantages of this disclosure more apparent and understandable, several embodiments are described in detail below, but they are not intended to limit the scope of this disclosure.

[0057] Example 1 - Sequence conservation analysis of primer pairs and probe combinations with database

[0058] The primers and probes were aligned with the database using NCBI's BLAST (Basic Local Alignment Search Tool). The sequences of the primers and probes were compared with the rhinovirus 5'UTR sequences (rhinovirus types A, B, and C) in the NCBI database (sampling time of the NCBI database was November 2024). Mismatches and sequence coverage (conservation) were analyzed. The results are shown in Table 1 below.

[0059] As shown in Table 1, compared with the sequence in the NCBI database, the sequence coverage of the forward primers, reverse primers, and probes (SEQ ID NOs: 2, 13, 14, 17) for detecting the 5'UTR of type A rhinovirus provided in this embodiment (under the condition of 1-mer mismatch) can reach more than 90%; the sequence coverage of the forward primers, reverse primers, and probes (SEQ ID NOs: 4, 6, 8, 14, 17) for detecting the 5'UTR of type B rhinovirus provided in this embodiment (under the condition of 1-mer mismatch) can reach more than 75%; and the sequence coverage of the forward primers, reverse primers, and probes (SEQ ID NOs: 2, 13, 14, 17) for detecting the 5'UTR of type C rhinovirus provided in this embodiment (under the condition of 1-mer mismatch) can reach more than 95%.

[0060] Example 2 - Screening of primer pairs and probe combinations against type A / C rhinovirus

[0061] Add 1x qP CR master mix (Biori 1X Neoscript RT premix, #FM5134), 500nM forward primer, 500nM reverse primer, and 100nM probe (SEQ ID NO:17) to deionized water to premix to form 20μL of reaction solution. RNA (RNVA / C ivtRNA, partial gene sequence of type A / C rhinovirus 5'UTR, SEQ ID NOs: 21, 22, with the aforementioned type A / C rhinovirus 5'UTR sequence introduced into pBluescript SK(+) plasmid, amplified using E. coli, purified, and then synthesized using a reverse transcription RNA synthesis kit) was serially diluted to concentrations of 0.2*10^6, 0.2*10^4, 0.2*10^2, and 0.2*10^1 copies / μL using 40 ng / μL tRNA.

[0062] The aforementioned premixed 20 μL reaction solution was transferred to a Bio-Rad 8-tube (#TLS-0801), and 5 μL of positive sample or 5 μL of 40 ng / μL tRNA (no template control (NTC)) for each condition was added to the 8-tube. After the addition was complete, the reaction tube was sealed with a Bio-Rad optical transparent cap (#TCS-0803).

[0063] Next, real-time quantitative reverse transcription polymerase chain reaction was performed using a Bio-Rad CFX Opus96 Real-Time PCR instrument (#12011319). Please refer to... Figure 2 , Figure 2 Display the temperature curve of the real-time quantitative reverse transcription polymerase chain reaction. For example... Figure 2 As shown, the reaction conditions were as follows: reverse transcription at 50°C for 5 minutes; enzyme activation at 95°C for 30 seconds; and PCR for 45 cycles, with denaturation at 95°C for 5 seconds and binding and amplification at 60°C for 10 seconds per cycle, and fluorescence detection at the end of each cycle.

[0064] After the reaction was completed, the cycle threshold (Cq value) was analyzed and statistically analyzed using CFX Maestro software. The results are shown in Table 2 below. RNV A1 / C and RNV A2 / C represent two different templates of type A and type C rhinovirus, respectively, containing the nucleotide sequences of SEQ ID NO:21 and SEQ ID NO:22.

[0065] Table 2

[0066] As shown in Table 2, the screening results of various combinations consisting of the forward primers shown in any of SEQ ID NOs:1-3, the reverse primers shown in any of SEQ ID NOs:14-16, and the probe shown in SEQ ID NO:17 are generally good. Among them, the detection results of groups 4 and 5, which can simultaneously detect RNV A1 / C and RNV A2 / C, are particularly good.

[0067] Example 3 - gDNA interference test of primer pairs and probe combinations against type A / C rhinovirus

[0068] First, human genomic DNA (gDNA) was prepared by extracting nucleic acids from fresh human whole blood using the Qiagen QIAamp DNABlood Mini kit (#51104). The extracted nucleic acid eluent was then quantified using the Qubit dsDNA HS Assay Kit (#032851).

[0069] Premix 1x qPCR master mix (Biori Neoscript RT Premix, #FM5134), 500 nM forward primer, 500 nM reverse primer, and 100 nM probe (SEQ ID NO: 17) with deionized water to prepare 15 μL of reaction solution. Then, add 200 ng of gDNA to the reaction solution to make the reaction volume 20 μL. Perform 10-fold serial dilutions of the artificially synthesized type A / C rhinovirus 5'UTR fragment template (SEQ ID NOs: 21, 22) as described above, and add 10^2 copies / μL of the template to 20 μL of reaction solution for each, making the final reaction volume 25 μL.

[0070] Real-time quantitative reverse transcription polymerase chain reaction was performed using a Bio-Rad CFX Opus96 Real-Time PCR instrument. For example... Figure 2 As shown, the reaction conditions were as follows: reverse transcription at 50°C for 5 minutes; enzyme activation at 95°C for 30 seconds; and PCR for 45 cycles, with denaturation at 95°C for 5 seconds and binding and amplification at 60°C for 10 seconds per cycle, and fluorescence detection at the end of each cycle.

[0071] After the reaction was completed, the cycle threshold (Cq value) was analyzed and statistically analyzed using CFX Maestro software. The results are shown in Table 3 below. RNV A1 / C and RNV A2 / C represent two different templates of rhinovirus type A and type C, respectively, containing the nucleotide sequences of SEQ ID NO:21 and SEQ ID NO:22.

[0072] As shown in Table 3, the primer pairs and probe combinations of groups 1, 2, 4, and 5 can identify A1 / C rhinovirus and are resistant to interference from high concentrations of human genomic DNA; the primer pairs and probe combinations of group 5 can identify A2 / C rhinovirus and are resistant to interference from high concentrations of human genomic DNA; and the primer pairs and probe combinations of group 5 (SEQ ID NOs: 2, 14, 17) can accurately identify both A1 / C and A2 / C rhinovirus simultaneously and are resistant to interference from high concentrations of human genomic DNA.

[0073] Example 4 - Screening of primer pairs and probe combinations against type B rhinovirus

[0074] Add 1x qP CR master mix (Biori 1X Neoscript RT premix, #FM5134), 500nM forward primer, 500nM reverse primer, and 100nM probe (SEQ ID NO:17) to deionized water to premix to form 20μL of reaction solution. The RNA (RNV B ivtRNA, whose full genome sequence of the B rhinovirus 5'UTR fragment template was outsourced for synthesis, SEQ ID NOs: 24, 25, and whose B rhinovirus 5'UTR sequence was introduced into pBluescript SK(+) plasmids, amplified using E. coli, purified, and then prepared as artificial RNA using a reverse transcription RNA synthesis kit) was serially diluted to concentrations of 0.2*10^6, 0.2*10^4, 0.2*10^2, and 0.2*10^1 copies / μL using 40 ng / μL tRNA.

[0075] The aforementioned premixed 20 μL reaction solution was transferred to a Bio-Rad 8-tube (#TLS-0801), and 5 μL of positive sample or 5 μL of 40 ng / μL tRNA (no template control (NTC)) for each condition was added to the 8-tube. After the addition was complete, the reaction tube was sealed with a Bio-Rad optical transparent cap (#TCS-0803).

[0076] Next, real-time quantitative reverse transcription polymerase chain reaction was performed using a Bio-Rad CFX Opus96 Real-Time PCR instrument (#12011319). Please refer to... Figure 2 , Figure 2 Display the temperature curve of the real-time quantitative reverse transcription polymerase chain reaction. For example... Figure 2 As shown, the reaction conditions were as follows: reverse transcription at 50°C for 5 minutes; enzyme activation at 95°C for 30 seconds; and PCR for 45 cycles, with denaturation at 95°C for 5 seconds and binding and amplification at 60°C for 10 seconds per cycle, and fluorescence detection at the end of each cycle.

[0077] After the reaction was completed, the cycle threshold (Cq value) was analyzed and statistically analyzed using CFX Maestro software. The results are shown in Table 4 below. RNV B1 and RNV B2 represent two different templates of type B rhinovirus, which contain the nucleotide sequences of SEQ ID NO:24 and SEQ ID NO:25, respectively.

[0078] Table 4

[0079] As shown in Table 4, the primer pairs and probe combinations of group 7 (SEQ ID NOs: 4, 14, 17) and group 11 (SEQ ID NOs: 5, 14, 17) showed better screening and detection results for RNV B1; the primer pairs and probe combinations of group 13 (SEQ ID NOs: 6, 14, 17) and group 15 (SEQ ID NOs: 12, 14, 17) showed better screening and detection results for RNV B2.

[0080] Example 5 - gDNA interference test of primer pairs and probe combinations against type B rhinovirus

[0081] First, human genomic DNA (gDNA) was prepared by extracting nucleic acids from fresh human whole blood using the Qiagen QIAamp DNABlood Mini kit (#51104). The extracted nucleic acid eluent was then quantified using the Qubit dsDNA HS Assay Kit (#032851).

[0082] Premix 1x qPCR master mix (Biori Neoscript RT Premix, #FM5134), 500 nM forward primer, 500 nM reverse primer, and 100 nM probe (SEQ ID NO: 17) with deionized water to prepare 15 μL of reaction solution. Then, add 200 ng of gDNA to the reaction solution to make the reaction volume 20 μL. Perform 10-fold serial dilutions of the artificially synthesized rhinovirus 5'UTR fragment template (SEQ ID NOs: 24, 25) as described above, and add 10^2 copies / μL of the template to 20 μL of reaction solution for each, making the final reaction volume 25 μL.

[0083] Real-time quantitative reverse transcription polymerase chain reaction was performed using a Bio-Rad CFX Opus96 Real-Time PCR instrument. For example... Figure 2 As shown, the reaction conditions were as follows: reverse transcription at 50°C for 5 minutes; enzyme activation at 95°C for 30 seconds; and PCR for 45 cycles, with denaturation at 95°C for 5 seconds and binding and amplification at 60°C for 10 seconds per cycle, and fluorescence detection at the end of each cycle.

[0084] After the reaction was completed, the cycle threshold (Cq value) was analyzed and statistically analyzed using CFX Maestro software. The results are shown in Table 5 below. RNV B1 and RNV B2 represent two different templates of rhinovirus type B, which contain the nucleotide sequences of SEQ ID NO:24 and SEQ ID NO:25, respectively.

[0085] Table 5

[0086] As shown in Table 5, the primer pairs and probe combinations of group 7 (SEQ ID NOs: 4, 14, 17) can accurately identify RNVB1 rhinovirus and are resistant to interference from high concentrations of human genomic DNA; the primer pairs and probe combinations of group 13 (SEQ ID NOs: 6, 14, 17) can accurately identify RNV B2 rhinovirus and are resistant to interference from high concentrations of human genomic DNA.

[0087] Example 6 - PCR efficiency test of primer pairs and probe combinations

[0088] 1x qPCR master mix (Biori Neoscript RT premix), 500 nM forward primer, 500 nM reverse primer, and 100 nM probe (a combination of primer pairs and probes from groups 5, 7, and 13) were premixed with deionized water to prepare 20 μL of reaction solution. Separately, synthetic RNAs (RNV A / C, RNV B ivtRNA) containing synthetic type A, type B, and type C rhinovirus 5'UTR fragment templates (SEQ IDNOs: 21, 22, 23, 24, 25) were serially diluted 10-fold, and templates of 10, 10^2, 10^3, 10^4, and 10^5 copies / μL were added to 20 μL of reaction solution, resulting in a final reaction volume of 25 μL.

[0089] Next, a real-time quantitative reverse transcription polymerase chain reaction was performed using a Bio-Rad CFX Opus96 Real-Time PCR instrument. For example... Figure 2 As shown, the reaction conditions were as follows: reverse transcription at 50°C for 5 minutes; enzyme activation at 95°C for 30 seconds; and PCR for 45 cycles, with denaturation at 95°C for 5 seconds and binding and amplification at 60°C for 10 seconds per cycle, and fluorescence detection at the end of each cycle.

[0090] After the reaction, the amplification curves obtained from the real-time quantitative reverse transcription polymerase chain reaction were analyzed using CFX Maestro software (e.g., Figures 3A to 3E As shown in the figure (using the results of a combination of groups 5, 7, and 13 as an example), the amplification curves show that the fluorescence values ​​of 10 to 10^6 copies all show a positive upward trend.

[0091] Furthermore, standard curves, Cq values, PCR efficiency (E%), R², and slope were analyzed and statistically analyzed using CFX Maestro software. The results are shown in Table 6 below.

[0092] Table 6

[0093] Based on the cycle thresholds (Cq values) obtained for different copy numbers, the detection range of the primer pairs and probe combinations designed in this disclosure can be verified. As shown in Table 6, real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed under different template amounts (10–10^6 copy numbers), and the R^2 values ​​obtained for different types of rhinoviruses were all greater than 0.97, indicating high accuracy of this regression model. Furthermore, the PCR efficiency of the primer pairs and probe combinations designed in this disclosure can reach over 95% for different types of rhinoviruses at various concentrations.

[0094] Example 7 - Product Analysis of Real-Time Quantitative Reverse Transcription Polymerase Chaining Reaction

[0095] First, human genomic DNA (gDNA) was prepared by extracting nucleic acids from fresh human whole blood using the Qiagen QIAamp DNABlood Mini kit (#51104). The extracted nucleic acid eluent was then quantified using the Qubit dsDNA HS Assay Kit (#032851).

[0096] 1x qPCR master mix (Biori Neoscript RT Premix, #FM5134), 500 nM forward primer, 500 nM reverse primer, and 100 nM probe (a combination of primer pairs and probes from groups 5, 7, and 13) were premixed with deionized water to prepare 20 μL of reaction solution. Separately, synthetic RNAs (RNV A / C, RNV B ivtRNA) containing synthetic type A, type B, and type C rhinovirus 5'UTR fragment templates (SEQ ID NOs: 21, 22, 23, 24, 25) were serially diluted 10-fold, and templates of 20, 10^2, and 10^3 copies / μL were added to 20 μL of reaction solution, resulting in a final reaction volume of 25 μL.

[0097] Real-time quantitative reverse transcription polymerase chain reaction was performed using a Bio-Rad CFX Opus96 Real-Time PCR instrument. For example... Figure 2 As shown, the reaction conditions were as follows: reverse transcription at 50°C for 5 minutes; enzyme activation at 95°C for 30 seconds; and PCR for 45 cycles, with denaturation at 95°C for 5 seconds and binding and amplification at 60°C for 10 seconds per cycle, and fluorescence detection at the end of each cycle.

[0098] After the reaction, the PCR products were diluted 10X with TE buffer and analyzed by capillary electrophoresis using an Agilent Fragment Analyzer 5300 (#M5311AA) and dsDNA 905Reagent (#DNF-905-K0500). Finally, fragment size analysis was performed using ProSize data analysis software. The results are as follows: Figure 4 As shown.

[0099] like Figure 4 As shown, from left to right, the columns represent: a marker ladder for molecular weight indication, template groups of 10^4, 20, and 10^2 copies / μL, a template group of 10^2 copies / μL plus 200 ng gDNA, and a template-free control group (NTC). Electrophoresis results show that the template groups of 10^4, 20, and 10^2 copies / μL, and the template group of 10^2 copies / μL plus 200 ng gDNA, all produced specific amplification products of rhinovirus (156 bp). Therefore, the primer pairs and probe combinations provided in this disclosure (a combination of groups 5, 7, and 13, SEQ ID NOs: 2, 4, 6, 8, 13, 14, and 17) can accurately identify rhinovirus types A, B, and C, and can withstand interference from high concentrations of human genomic DNA.

[0100] Example 8 - Analysis of Lyophilized Dosage Forms of Primer Pairs and Probe Combinations

[0101] 1x qPCR master mix, 500nM forward primer, 500nM reverse primer, and 100nM probe were premixed with deionized water to form a 20μL reaction solution, which was then divided into two groups: one for liquid reagents and the other for lyophilized reagents. The lyophilized reagents were prepared by sublimation at -40℃, -20℃, 0℃, and 25℃ under near-vacuum conditions.

[0102] Additionally, the RNV RNA (RNV A1 / C, A2 / C, A3 / C, B1, B2 ivtRNA) template containing the aforementioned synthetic rhinovirus 5'UTR fragment was serially diluted 10-fold. 5 μL of RNV RNA templates at 10, 10^2, 10^3, 10^4, and 10^5 copies / μL were added to the reaction solutions of the aforementioned 20 μL liquid reagent group and lyophilized reagent group, making the final reaction volume 25 μL.

[0103] Real-time quantitative reverse transcription polymerase chain reaction was performed using a Bio-Rad CFX Opus96 Real-Time PCR instrument. For example... Figure 2 As shown, the reaction conditions were as follows: reverse transcription at 50°C for 5 minutes; enzyme activation at 95°C for 30 seconds; and PCR for 45 cycles, with denaturation at 95°C for 5 seconds and binding and amplification at 60°C for 10 seconds per cycle, and fluorescence detection at the end of each cycle.

[0104] After the reaction was completed, the cycle threshold (Cq value) was analyzed and statistically analyzed using CFX Maestro software. The results are shown in Table 7 below.

[0105] Table 7

[0106] As shown above, the freeze-dried primer pairs and probe combinations, after being reconstituted in water, can achieve the same efficiency as liquid reagents by performing real-time quantitative polymerase chain reactions.

[0107] Example 9 - Limit of Detection (LoD) Analysis of Primer Pairs and Probe Combinations

[0108] 1x qPCR master mix, 500nM forward primer, 500nM reverse primer, and 100nM probe were premixed with deionized water to form a 20μL reaction solution, which was then dried using a lyophilization program to prepare lyophilized drug cakes.

[0109] 20 μL of TF-1a human erythroblasts (Leadgene, 10^6 cells / mL) were premixed with 380 μL of Copan Universal Transport Medium (#330C). Extraction was performed using the Dagene G1 fully automated nucleic acid detection system. First, 400 μL of the premixed solution was added to Proteinase K for the extraction cartridge, followed by 400 μL of Lysis buffer. All the mixture was then transferred to the extraction cartridge and placed in the instrument for extraction. After the extraction process, the eluent was collected for later use.

[0110] The lyophilized drug cake was dissolved in 20 μL of the previously collected extraction eluent. Then, the RNV RNA (RNV A1 / C, A2 / C, A3 / C, B1, B2 ivtRNA) containing the aforementioned synthetic rhinovirus 5'UTR fragment template was serially diluted to 0.2*10^2 and 0.2*10^1 copies / μL using tRNA at a concentration of 40 ng / μL.

[0111] Add 20 μL of the reaction reagent dissolved in the lyophilized drug cake to a G1-specific reaction tube, then add 5 μL of the positive sample from each of the aforementioned conditions for mixing, or add 5 μL of 40 ng / μL tRNA from the NTC group to the G1 reaction tube. After adding, seal the G1 reaction tube with the cap.

[0112] The reaction was performed using the Dagene G1 fully automated nucleic acid detection system. The reaction conditions were as follows: reverse transcription at 50°C for 5 minutes; enzyme activation at 95°C for 30 seconds; and PCR for 45 cycles, with denaturation at 95°C for 5 seconds and binding and amplification at 60°C for 10 seconds per cycle. Fluorescence detection was performed at the end of each cycle.

[0113] After the reaction was completed, the Cq values ​​displayed by the Dagene G1 fully automated nucleic acid detection system were analyzed and statistically analyzed. The results are shown in Table 8 below.

[0114] Table 8

[0115] As shown in Table 8, under the condition of a high concentration of internal control (IC), the detection limit (LoD) of the primer pairs and probe combinations provided in this embodiment can be less than 20 copies / rxn.

[0116] Example 10 - Sensitivity Analysis of Primer Pairs and Probe Combinations

[0117] First, human genomic DNA (gDNA) was prepared by extracting nucleic acids from fresh human whole blood using the Qiagen QIAamp DNABlood Mini kit (#51104). The extracted nucleic acid eluent was then quantified using the Qubit dsDNA HS Assay Kit (#032851).

[0118] 1x qPCR master mix (Biori Neoscript RT Premix, #FM5134), 500 nM forward primer, 500 nM reverse primer, and 100 nM probe (a combination of primer pairs and probes from groups 5, 7, and 13) were premixed with deionized water to prepare 15 μL of reaction solution. Next, 200 ng of gDNA was added to the reaction solution to bring the total reaction volume to 20 μL. Synthetic RNAs (RNV A / C, RNV B ivtRNA) containing synthetic A, B, and C rhinovirus 5'UTR fragment templates (SEQ ID NOs: 21, 22, 23, 24, 25) were serially diluted 10-fold, and 10^2 copies / μL of template and 200 ng of gDNA were each added to 20 μL of reaction solution, bringing the final reaction volume to 25 μL.

[0119] Real-time quantitative reverse transcription polymerase chain reaction was performed using a Bio-Rad CFX Opus96 Real-Time PCR instrument. For example... Figure 2 As shown, the reaction conditions were as follows: reverse transcription at 50°C for 5 minutes; enzyme activation at 95°C for 30 seconds; and PCR for 45 cycles, with denaturation at 95°C for 5 seconds and binding and amplification at 60°C for 10 seconds per cycle, and fluorescence detection at the end of each cycle.

[0120] Furthermore, the Cq value was calculated using CFX Maestro software, and the results are shown in Table 9 below.

[0121] Table 9

[0122] As shown in Table 9, the primer pairs and probe combinations provided in this disclosure (combinations of primer pairs and probes from groups 5, 7, and 13, SEQ ID NOs: 2, 4, 6, 8, 13, 14, and 17) can also detect 100 copies / rxn under interference from 200 ng gDNA, indicating that they can perform highly sensitive detection of rhinovirus under interference from high concentrations of human genomic DNA.

[0123] Example 11 - Detection results of primer pairs and probe combinations on inactivated rhinovirus strains

[0124] 1x qPCR master mix, 500nM forward primer, 500nM reverse primer, and 100nM probe (a combination of primer pairs and probes from groups 5, 7, and 13) were premixed with deionized water to prepare a 20μL reaction solution, which was then dried using a lyophilization program.

[0125] NATtrol with PBS buffer TM The Rhinovirus (strain 1A) in Respiratory Verification Panel 2.1 is diluted to a concentration of 100, 10 copies / μL.

[0126] The entire analysis process (nucleic acid extraction + polymerase chain reaction) was performed using the Dagene G1 fully automated nucleic acid detection system. First, the aforementioned dried reagents were loaded into the cartridge. Then, 380 μL of Copan Universal Transport Medium (#330C) was mixed with 20 μL of virus solution prepared in the previous step to achieve a final sample concentration of 5000 and 500 copies / mL. Next, Proteinase K for extraction cartridges was added, followed by 400 μL of Lysis buffer. All the mixture was then transferred to the cartridge and placed into the instrument for the entire process.

[0127] The reaction was performed using the Dagene G1 fully automated nucleic acid detection system. The reaction conditions were as follows: reverse transcription at 50°C for 5 minutes; enzyme activation at 95°C for 30 seconds; and PCR for 45 cycles, with denaturation at 95°C for 5 seconds and binding and amplification at 60°C for 10 seconds per cycle. Fluorescence detection was performed at the end of each cycle.

[0128] After the reaction was completed, the Cq values ​​displayed by the Dagene G1 fully automated nucleic acid detection system were analyzed and statistically analyzed. The results are shown in Table 10 below.

[0129] Table 10

[0130] As shown in Table 10, the rhinovirus RNV 1A strain can be detected by using the Dagene G1 instrument with the primer pairs and probe combinations provided in the present invention to detect inactivated rhinovirus strains.

[0131] Example 12 - Detection results of primer pairs and probe combinations for rhinovirus and comparison with commercially available products

[0132] The lyophilized reagent was prepared according to the method in Example 8 (using RNV A1 / C RNA template), the lyophilized reagent was reconstituted with pure water, and then rhinovirus RNA (NATtrol) was added separately. TMRhinovirus (strain 1A) in Respiratory Verification Panel 2.1 was extracted using the TANBead Nucleic Acid Extraction Kit OptiPure ViralAuto Tube (M665S46) on a TANBead Maelstrom 4810 instrument, following the instructions provided to obtain an extract containing rhinovirus RNA. The primer pairs and probe combinations provided in this embodiment were compared with the commercially available Certest VIASURE Rhinovirus+Enterovirus Kit (hereinafter referred to as the Certest kit). The Certest kit was operated according to its instructions, and the aforementioned rhinovirus RNA template was added.

[0133] Real-time quantitative reverse transcription polymerase chain reaction was performed using a Bio-Rad CFX Opus 96 Real-Time PCR instrument. For example... Figure 2 As shown, the reaction conditions were as follows: reverse transcription at 50℃ for 5 minutes; enzyme activation at 95℃ for 30 seconds; and PCR for 45 cycles, with denaturation at 95℃ for 5 seconds and binding and amplification at 60℃ for 10 seconds per cycle, and fluorescence detection at the end of each cycle. After the reaction, the detection limits (LoD) of the primer pairs and probe combinations provided in this embodiment (combinations of primer pairs and probes from groups 5, 7, and 13, SEQ ID NOs: 2, 4, 6, 8, 13, 14, and 17) and the commercially available product Certest lit were determined using the Dagene G1 fully automated nucleic acid detection system software. The LoD was determined based on the lowest concentration that could be stably detected. The results are shown in Table 11.

[0134] Table 11

[0135] As shown in Table 11, the rhinovirus test kit provided in this embodiment can detect A / C type rhinovirus strains, and the detection limit for rhinovirus can reach 30 copies / rxn, which is superior to Certest kit.

[0136] Example 13 - Cross-reactivity analysis of primer pairs and probe combinations

[0137] Using ZeptoMetrix NATtrol TMThe Respiratory Verification Panel 2.1 serves as a standard, which is a deactivated pathogen solution. Extraction was performed using the TANBead Nucleic Acid Extraction Kit OptiPure ViralAuto Tube (M665S46) on a TANBead Maelstrom 4810 machine. Each pathogen was extracted separately, following the instructions provided in the package, to obtain an extract containing the pathogen's nucleic acid.

[0138] Premix 1x qPCR master mix (Biori 1X Neoscript RT premix, #FM5134), 500nM forward primer, 500nM reverse primer, and 100nM probe with deionized water to prepare a 20μL reaction solution. Add 5μL of the aforementioned nucleic acid extraction product to each of the 20μL reaction solutions to make a final reaction volume of 25μL.

[0139] Next, real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed using a Bio-Rad CFX Opus96 Real-Time PCR instrument. The reaction conditions were: reverse transcription at 50°C for 5 minutes; enzyme activation at 95°C for 30 seconds; and 45 cycles of PCR, each cycle consisting of 5 seconds of denaturation at 95°C and 10 seconds of binding and amplification at 60°C, with fluorescence detection performed at the end of each cycle. After the reaction, the Bio-Rad CFX Maestro software was used to determine whether the reaction was positive or negative. The results are shown in Table 12 below.

[0140] Table 12

[0141] As shown in Table 12, the rhinovirus test kit provided in this disclosure embodiment has no cross-reactivity with other common respiratory pathogens.

[0142] In summary, according to the embodiments of this disclosure, a primer pair, a kit, and a method for detecting rhinovirus are provided. The primer pair targets specific gene fragments of rhinovirus, and performs highly sensitive, highly specific, and rapid detection with these specific gene fragments as the amplification target. According to the embodiments of this disclosure, the kit for detecting rhinovirus can withstand interference from high concentrations of human genomic DNA, maintain the detection sensitivity of polymerase chain reaction, and maintain a specific gene fragment sequence coverage of more than 75% for the three different rhinoviruses: types A, B, and C.

[0143] While the embodiments and advantages of this disclosure have been presented above, it should be understood that anyone skilled in the art can make modifications, substitutions, and refinements without departing from the spirit and scope of this disclosure. Furthermore, each claim constitutes an individual embodiment, and the scope of protection of this disclosure also includes combinations of the various claims and embodiments. The scope of protection of this disclosure shall be determined by the appended claims.

Claims

1. A primer pair for detecting rhinovirus, comprising a forward primer and a reverse primer targeting a 5' untranslated region (5'UTR), wherein the 5'UTR comprises a nucleotide sequence as shown in any of S EQ ID NOs:21 to 25; in, The forward primer is 12 to 30 nucleotides in length and corresponds to the nucleotide sequence between sites 1 and 70 of any of the SE QID NOs: 21 to 25; and The reverse primer is 12 to 30 nucleotides in length and is a nucleotide sequence complementary to the nucleotide sequence between the 180th and 204th sites shown in any of the SE QID NOs:21 to 25.

2. The primer pair for detecting rhinovirus as described in claim 1, wherein the length of the forward primer is 14 to 22 nucleotides, and / or the length of the reverse primer is 16 to 23 nucleotides.

3. The primer pair for detecting rhinovirus as described in claim 1, wherein the forward primer comprises a nucleotide sequence as shown in any one of SE QID N Os:1 to 13.

4. The primer pair for detecting rhinovirus as claimed in claim 1, wherein the reverse primer comprises a nucleotide sequence as shown in any one of SE QID N Os:14 to 16.

5. A kit for detecting rhinovirus, comprising: A forward primer, a reverse primer, and a probe, wherein the forward primer, the reverse primer, and the probe target the 5' untranslated region (5'UTR), and the 5'UTR comprises a nucleotide sequence as shown in any of SE QID NOS:21 to 25; The forward primer is 12 to 30 nucleotides in length and corresponds to the nucleotide sequence between sites 1 and 70 of any of SE QID NOS:21 to 25; and The reverse primer is 12 to 30 nucleotides in length and is a nucleotide sequence complementary to the nucleotide sequence between the 180th and 204th sites shown in any of SE QID NO S:21 to 25.

6. The kit for detecting rhinovirus as described in claim 5, wherein the length of the forward primer is 14 to 22 nucleotides, and / or the length of the reverse primer is 16 to 23 nucleotides.

7. The kit for detecting rhinovirus as claimed in claim 5, wherein the forward primer comprises a nucleotide sequence as shown in any one of SE QID N Os:1 to 13.

8. The kit for detecting rhinovirus as claimed in claim 5, wherein the reverse primer comprises a nucleotide sequence as shown in any one of SE QID N Os:14 to 16.

9. The kit for detecting rhinovirus as described in claim 5, wherein the probe is 13 to 21 nucleotides in length.

10. The kit for detecting rhinovirus as claimed in claim 5, wherein the probe comprises a nucleotide sequence as shown in SE QID NO:

17.

11. The kit for detecting rhinovirus as claimed in claim 5, wherein the 5' end of the probe is connected to a reporter dye and the 3' end of the probe is connected to a quencher.

12. The kit for detecting rhinovirus as claimed in claim 5, further comprising a forward primer, a reverse primer, and a probe targeting a nucleic acid sequence of a human gene, wherein the forward primer comprises a nucleotide sequence as shown in SE QID NO:18, the reverse primer comprises a nucleotide sequence as shown in SE QID NO:19, and the probe comprises a nucleotide sequence as shown in SE QID NO:

20.

13. A method for detecting rhinovirus, comprising the following steps: Provide one copy; Provide a primer pair comprising a forward primer and a reverse primer targeting the 5' untranslated region (5'UTR), wherein the 5'UTR comprises a nucleotide sequence as shown in any of SE QID NOs:21 to 25; in, The forward primer is 12 to 30 nucleotides in length and corresponds to the nucleotide sequence between sites 1 and 70 of any of the SE QID NOs: 21 to 25; and The reverse primer is 12 to 30 nucleotides in length and is a nucleotide sequence complementary to the nucleotide sequence between the 180th and 204th sites shown in any of SE QID NO S:21 to 25; The primer pair was used to perform a polymerase chain reaction with the sample to obtain the product; as well as The product was analyzed to detect the presence of rhinovirus.

14. The method for detecting rhinovirus as claimed in claim 13, further comprising providing a probe, wherein the probe is 13 to 21 nucleotides in length and is a nucleotide sequence corresponding to the 83rd to 104th sites shown in any one of SE QID NOs:21 to 25.

15. The method for detecting rhinovirus as described in claim 14, wherein the probe comprises a nucleotide sequence as shown in SE QID NO:

17.

16. The method for detecting rhinovirus as claimed in claim 14, wherein the 5' end of the probe is connected to a reporter dye and the 3' end of the probe is connected to a quencher.

17. The method for detecting rhinovirus as described in claim 13, wherein the sample source includes saliva samples, sputum samples, nasal swab samples, throat swab samples, nasopharyngeal samples, or body fluid samples.

18. The method for detecting rhinovirus as claimed in claim 13, wherein the step of using the primer pair to perform the polymerase cascade reaction with the sample to obtain the product includes performing a real-time quantitative reverse transcription polymerase cascade reaction to amplify the nucleotide sequence of the 5'UTR in the rhinovirus by the primer pair to obtain the product.

19. The method for detecting rhinovirus as claimed in claim 13, further comprising providing a forward primer, a reverse primer, and a probe for a nucleic acid sequence of a human gene, wherein the forward primer comprises a nucleotide sequence as shown in SE QID NO:18, the reverse primer comprises a nucleotide sequence as shown in SE QID NO:19, and the probe comprises a nucleotide sequence as shown in SE QID NO:20.

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