Pathogen-specific probe set for simultaneously detecting pathogens causing respiratory syndrome and use thereof

Abstract

The present invention relates to a pathogen-specific probe set capable of simultaneously detecting viruses and bacteria associated with respiratory syndromes, a composition for diagnosing respiratory syndrome comprising same, a kit for diagnosing respiratory syndrome comprising same, and a method for simultaneously detecting respiratory syndrome-associated viruses and bacteria using the probe set.

Description

Pathogen-specific probe set for simultaneous detection of respiratory syndrome pathogens and its use

[0001] The present invention relates to a pathogen-specific probe set for simultaneous detection of respiratory syndrome pathogens and its use.

[0002] Respiratory diseases caused by viruses are among the most prevalent diseases, accounting for approximately half of all infectious disease outbreaks.

[0003] Respiratory diseases are pathological conditions that occur in the organs and tissues, making gas exchange difficult, and refer to diseases that occur in the state of all respiratory organs, including the trachea, bronchi, bronchioles, alveoli, pleural pleural space, respiratory muscles, and nerves.

[0004] They range from mild illnesses such as colds, influenza, and pharyngitis to life-threatening conditions such as bacterial pneumonia, pulmonary embolism, tuberculosis, acute asthma, lung cancer, and severe acute respiratory syndrome.

[0005] Among the statutory infectious diseases, nine respiratory infection-causing pathogens and their diseases are designated as Class 4 statutory infectious diseases and are managed accordingly.

[0006] When respiratory illnesses caused by infectious diseases occur, existing diagnostic methods can be used to diagnose them. However, when a new or unknown infectious disease is suspected, diagnostic methods that can confirm the causative agent, along with exclusionary diagnosis based on existing pathogen information, are necessary. Furthermore, quickly identifying the pathogen causing the disease is crucial for early detection and early response. However, technological limitations make it difficult to efficiently screen for and diagnose multiple pathogens simultaneously.

[0007] To overcome these limitations, we will utilize a NGS method that is differentiated from the existing method to produce a multi-test panel, and through rapid pathogen screening, we will accurately identify and exclude the causative pathogen, which will help in future responses.

[0008] The present invention was derived from the above-mentioned needs, and the inventors of the present invention have completed the present invention by identifying a probe set capable of simultaneously detecting viruses and bacteria based on respiratory syndrome, a composition for diagnosing respiratory syndrome, and a method for simultaneously detecting viruses and bacteria based on respiratory syndrome using the same.

[0009] In order to solve the above problem, the present invention provides a probe set for simultaneous detection of respiratory syndrome pathogens comprising SEQ ID NO: 1 to SEQ ID NO: 37966, wherein SEQ ID NO: 1 to 433 are for detecting Bacillus anthracis, SEQ ID NO: 434 to 766 are for detecting Blastomyces dermatitidis, SEQ ID NO: 767 to 1122 are for detecting Bordetella pertussis, SEQ ID NO: 1123 to 1490 are for detecting Chlamydia pneumoniae, SEQ ID NO: 1491 to 1863 are for detecting Chlamydia psittaci, and SEQ ID NO: 1864 to 2266 are for detecting Coccidioides immitis. and SEQ ID NOs: 2267 to 2665 are for detecting Coccidioides posadasii, SEQ ID NOs: 2666 to 3022 are for detecting Corynebacterium diphtheria, SEQ ID NOs: 3023 to 3409 are for detecting Coxiella burnetii, SEQ ID NOs: 3410 to 3778 are for detecting Francisella tularensis, SEQ ID NOs: 3779 to 4155 are for detecting Haemophilus influenzae, SEQ ID NOs: 4156 to 4544 are for detecting Histoplasma capsulatum, and SEQ ID NOs: 4545 to 4912 are for detecting Legionella. It is for the detection of Legionella pneumophila, and sequence numbers 4913 to 5318 are for the detection of Mycobacterium avium.SEQ ID NOs: 5319 to 5734 are for detecting Mycobacterium intracellulare, SEQ ID NOs: 5735 to 6230 are for detecting Mycobacterium kansassi, SEQ ID NOs: 6231 to 6652 are for detecting Mycobacterium tuberculosis, SEQ ID NOs: 6653 to 7070 are for detecting Mycobacterium abscessus, SEQ ID NOs: 7071 to 7421 are for detecting Mycoplasmoides pneumoniae, SEQ ID NOs: 7422 to 7784 are for detecting Neisseria meningitidis, and SEQ ID NOs: 7785 to 8168 are for detecting For the detection of Streptococcus pneumoniae, SEQ ID NOs: 8169 to 8539 are for the detection of Streptococcus pyogenes, SEQ ID NOs: 8540 to 8978 are for the detection of Yersinia pestis, SEQ ID NOs: 8979 to 9441 are for the detection of Coronavirus 229E, SEQ ID NOs: 9442 to 9935 are for the detection of Coronavirus HKU1, SEQ ID NOs: 9936 to 10394 are for the detection of Coronavirus NL63, SEQ ID NOs: 10395 to 10906 are for the detection of Coronavirus OC43, and SEQ ID NOs: 10907 to 14833 are It is for the detection of cytomegalovirus, and sequence numbers 14834 to 17628 are for the detection of Epstein-Barr virus.SEQ ID NOs: 17629 to 17827 are for detecting Hantavirus, SEQ ID NOs: 17828 to 18130 are for detecting Hendravirus, SEQ ID NOs: 18131 to 18432 are for detecting Henipavirus, SEQ ID NOs: 18433 to 19011 are for detecting Human adenovirus type 14, SEQ ID NOs: 19012 to 19610 are for detecting Human adenovirus type 1, SEQ ID NOs: 19611 to 20199 are for detecting Human adenovirus type 21, and SEQ ID NOs: 20200 to 20797 are for detecting Human adenovirus type 2. 2) For detection, SEQ ID NOs: 20798 to 21384 are for detection of human adenovirus type 3, SEQ ID NOs: 21385 to 21983 are for detection of human adenovirus type 4, SEQ ID NOs: 21984 to 22562 are for detection of human adenovirus type 55, SEQ ID NOs: 22563 to 23160 are for detection of human adenovirus type 5, SEQ ID NOs: 23161 to 23747 are for detection of human adenovirus type 7, SEQ ID NOs: 23748 to 24330 are for detection of human adenovirus type 8, SEQ ID NOs: 24331 to 24898 are for detecting human adenovirus type,Sequence numbers 24899 to 24989 are for detecting human bocavirus 1, sequence numbers 24990 to 25074 are for detecting human bocavirus 2, sequence numbers 25075 to 25161 are for detecting human bocavirus 3, sequence numbers 25162 to 25246 are for detecting human bocavirus 4, sequence numbers 25247 to 25369 are for detecting human enterovirus CV-A21, sequence numbers 25370 to 25492 are for detecting human enterovirus EV-A71, and sequence numbers 25493 to 25615 is for detecting human enterovirus EV-C104, SEQ ID NOs: 25616 to 25736 are for detecting human enterovirus EV-C105, SEQ ID NOs: 25737 to 25858 are for detecting human enterovirus EV-C109, SEQ ID NOs: 25859 to 25980 are for detecting human enterovirus EV-C117, SEQ ID NOs: 25981 to 26102 are for detecting human enterovirus EV-C118, and SEQ ID NOs: 26103 to 26224 are for detecting human enterovirus EV-D68. enterovirus EV-D68) and sequence numbers 26225 to 28915 are for the detection of human herpesvirus 6.Sequence numbers 28916 to 29137 are for detecting human metapneumovirus A, sequence numbers 29138 to 29358 are for detecting human metapneumovirus B, sequence numbers 29359 to 29480 are for detecting human parechovirus, sequence numbers 29481 to 29695 are for detecting influenza virus A-H1N1, sequence numbers 29696 to 29909 are for detecting influenza virus A-H3N2, sequence numbers 29910 to 30131 are for detecting influenza virus A, and sequence numbers 30132 to 30320 is for detecting Influenza virus B-victoria, SEQ ID NOs: 30321 to 30556 are for detecting Influenza virus B, SEQ ID NOs: 30557 to 30774 are for detecting Influenza virus B-yamagata, SEQ ID NOs: 30775 to 30986 are for detecting Influenza virus C, SEQ ID NOs: 30987 to 31196 are for detecting Influenza virus D, SEQ ID NOs: 31197 to 31460 are for detecting Measles morbillivirus, and SEQ ID NOs: 31461 to 31962 are It is for the detection of MERS-CoV, and sequence numbers 31963 to 32218 are for the detection of mumps orthorubulavirus.Sequence numbers 32219 to 32522 are for detecting Nipahvirus, sequence numbers 32523 to 32781 are for detecting Parainfluenza virus 1, sequence numbers 32782 to 33041 are for detecting Parainfluenza virus 2, sequence numbers 33042 to 33298 are for detecting Parainfluenza virus 3, sequence numbers 33299 to 33582 are for detecting Parainfluenza virus 4a, sequence numbers 33583 to 33868 are for detecting Parainfluenza virus 4b, and sequence numbers 33869 to 34121 is for the detection of Respiratory Syncytial virus A, SEQ ID NOs: 34122 to 34374 are for the detection of Respiratory Syncytial virus B, SEQ ID NOs: 34375 to 34493 are for the detection of Rhinovirus A, SEQ ID NOs: 34494 to 34614 are for the detection of Rhinovirus B, SEQ ID NOs: 34615 to 34732 are for the detection of Rhinovirus C, SEQ ID NOs: 34733 to 34894 are for the detection of Rubivirus rubellae, SEQ ID NOs: 34895 to 35392 are for the detection of SARS-CoV-2, and SEQ ID NOs: 35393 to 35887 is for the detection of SARS-CoV, and sequence numbers 35888 to 37966 are for the detection of Varicella zoster virus.Provides a probe set for simultaneous detection of respiratory syndrome pathogens.

[0010] The above probe set can be prepared by isolating bacteria or viruses, and the probes included in the above probe set are SEQ ID NOs: 1 to 433; SEQ ID NOs: 434 to 766; SEQ ID NOs: 767 to 1122; SEQ ID NOs: 1123 to 1490; SEQ ID NOs: 1491 to 1863; SEQ ID NOs: 1864 to 2266; SEQ ID NOs: 2267 to 2665; SEQ ID NOs: 2666 to 3022; SEQ ID NOs: 3023 to 3409; SEQ ID NOs: 3410 to 3778; SEQ ID NOs: 3779 to 4155; SEQ ID NOs: 4156 to 4544; SEQ ID NOs: 4545 to 4912; SEQ ID NOs: 4913 to 5318; SEQ ID NOs: 5319 to 5734; SEQ ID NOs: 5735 to 6230; SEQ ID NOs: 6231 to 6652; SEQ ID NOs: 6653 to 7070; SEQ ID NOs: 7071 to 7421; SEQ ID NOs: 7422 to 7784; SEQ ID NOs: 7785 to 8168; SEQ ID NOs: 8169 to 8539; and SEQ ID NOs: 8540 to 8978, or SEQ ID NOs: 8979 to 9441; SEQ ID NOs: 9442 to 9935; SEQ ID NOs: 9936 to 10394; SEQ ID NOs: 10395 to 10906; SEQ ID NOs: 10907 to 14833; SEQ ID NOs: 14834 to 17628; SEQ ID NOs: 17629 to 17827; SEQ ID NOs: 17828 to 18130; SEQ ID NOs: 18131 to 18432; SEQ ID NOs: 18433 to 19011; SEQ ID NOs: 19012 to 19610; SEQ ID NOs: 19611 to 20199; SEQ ID NOs: 20200 to 20797; SEQ ID NOs: 20798 to 21384; SEQ ID NOs: 21385 to 21983; SEQ ID NOs: 21984 to 22562; SEQ ID NOs: 22563 to 23160; SEQ ID NOs: 23161 to 23747; SEQ ID NOs: 23748 to 24330; SEQ ID NOs: 24331 to 24898; SEQ ID NOs: 24899 to 24989; SEQ ID NOs: 24990 to 25074; SEQ ID NOs: 25075 to 25161; SEQ ID NOs: 25162 to 25246;SEQ ID NOs: 25247 to 25369; SEQ ID NOs: 25370 to 25492; SEQ ID NOs: 25493 to 25615; SEQ ID NOs: 25616 to 25736; SEQ ID NOs: 25737 to 25858; SEQ ID NOs: 25859 to 25980; SEQ ID NOs: 25981 to 26102; SEQ ID NOs: 26103 to 26224; SEQ ID NOs: 26225 to 28915; SEQ ID NOs: 28916 to 29137; SEQ ID NOs: 29138 to 29358; SEQ ID NOs: 29359 to 29480; SEQ ID NOs: 29481 to 29695; SEQ ID NOs: 29696 to 29909; SEQ ID NOs: 29910 to 30131; SEQ ID NOs: 30132 to 30320; SEQ ID NOs: 30321 to 30556; SEQ ID NOs: 30557 to 30774; SEQ ID NOs: 30775 to 30986; SEQ ID NOs: 30987 to 31196; SEQ ID NOs: 31197 to 31460; SEQ ID NOs: 31461 to 31962; SEQ ID NOs: 31963 to 32218; SEQ ID NOs: 32219 to 32522; SEQ ID NOs: 32523 to 32781; SEQ ID NOs: 32782 to 33041; SEQ ID NOs: 33042 to 33298; SEQ ID NOs: 33299 to 33582; SEQ ID NOs: 33583 to 33868; SEQ ID NOs: 33869 to 34121; It may include SEQ ID NOs: 34122 to 34374; SEQ ID NOs: 34375 to 34493; SEQ ID NOs: 34494 to 34614; SEQ ID NOs: 34615 to 34732; SEQ ID NOs: 34733 to 34894; SEQ ID NOs: 34895 to 35392; SEQ ID NOs: 35393 to 35887; and SEQ ID NOs: 35888 to 37966, and preferably SEQ ID NOs: 1 to 433; SEQ ID NOs: 767 to 1122; SEQ ID NOs: 1491 to 1863; SEQ ID NOs: 2666 to 3022; SEQ ID NOs: 3023 to 3409; SEQ ID NOs: 3410 to 3778; SEQ ID NOs: 3779 to 4155; SEQ ID NOs: 4545 to 4912; Sequence numbers 6231 to 6652;SEQ ID NOs: 7071 to 7421; SEQ ID NOs: 7422 to 7784; SEQ ID NOs: 7785 to 8168; SEQ ID NOs: 8169 to 8539; and SEQ ID NOs: 8540 to 8978, or SEQ ID NOs: 8979 to 9441; SEQ ID NOs: 9936 to 10394; SEQ ID NOs: 10395 to 10906; SEQ ID NOs: 10907 to 14833; SEQ ID NOs: 17629 to 17827; SEQ ID NOs: 19012 to 19610; SEQ ID NOs: 20200 to 20797; SEQ ID NOs: 20798 to 21384; SEQ ID NOs: 21385 to 21983; SEQ ID NOs: 21984 to 22562; SEQ ID NOs: 22563 to 23160; SEQ ID NOs: 23161 to 23747; SEQ ID NOs: 23748 to 24330; SEQ ID NOs: 24331 to 24898; SEQ ID NOs: 24899 to 24989; SEQ ID NOs: 25370 to 25492; SEQ ID NOs: 26103 to 26224; SEQ ID NOs: 29138 to 29358; SEQ ID NOs: 29359 to 29480; SEQ ID NOs: 29481 to 29695; SEQ ID NOs: 29696 to 29909; SEQ ID NOs: 30132 to 30320; SEQ ID NOs: 30321 to 30556; SEQ ID NOs: 30557 to 30774; SEQ ID NOs: 31197 to 31460; SEQ ID NOs: 31461 to 31962; SEQ ID NOs: 31963 to 32218; SEQ ID NOs: 32523 to 32781; SEQ ID NOs: 32782 to 33041; SEQ ID NOs: 33042 to 33298; SEQ ID NOs: 33299 to 33582; SEQ ID NOs: 33583 to 33868; SEQ ID NOs: 33869 to 34121; SEQ ID NOs: 34122 to 34374; SEQ ID NOs: 34375 to 34493; SEQ ID NOs: 34494 to 34614; SEQ ID NOs: 34615 to 34732; SEQ ID NOs: 34733 to 34894; SEQ ID NOs: 34895 to 35392; SEQ ID NOs: 35393 to 35887; and sequence numbers 35888 to 37966, but are not limited thereto.;

[0011] The concentration of the above probes can be selected and used in various ways by a person skilled in the art depending on the experimental conditions, and is preferably 1 to 1000 nM each, and more preferably 100 to 500 nM each, but is not limited thereto.

[0012] The present invention provides a probe set for simultaneous detection of respiratory syndrome pathogens, including Bacillus anthracis, Blastomyces dermatitidis, Bordetella pertussis, Chlamydia pneumoniae, Chlamydia psittaci, Coccidioides immitis, Coccidioides posadasii, Corynebacterium diphtheria, Coxiella burnetii, Francisella tularensis, Haemophilus influenzae, Histoplasma capsulatum, Legionella pneumophila, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansassi, Mycobacterium tuberculosis, Mycobacterium abscessus, Mycoplasmoides pneumoniae, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes, Yersinia pestis, Coronavirus 229E, Coronavirus HKU1, Coronavirus NL63, Coronavirus OC43, Cytomegalovirus,Epstein-Barr virus, Hantavirus, Hendravirus, Henipavirus, Human adenovirus type 14, Human adenovirus type 1, Human adenovirus type 21, Human adenovirus type 2, Human adenovirus type 3, Human adenovirus type 4, Human adenovirus type 55, Human adenovirus type 5, Human adenovirus type 7, Human adenovirus type 8, Human adenovirus type, Human bocavirus 1, Human bocavirus 2, Human bocavirus 3, Human bocavirus 4, Human enterovirus CV-A21, Human enterovirus EV-A71, Human enterovirus EV-C104, Human enterovirus EV-C105, Human enterovirus EV-C105, Human enterovirus EV-C109, Human enterovirus EV-C117, Human enterovirus EV-C118,Human enterovirus EV-D68, Human herpesvirus 6, Human metapneumovirus A, Human metapneumovirus B, Human parechovirus, Influenza virus A-H1N1, Influenza virus A-H3N2, Influenza virus A, Influenza virus B-victoria, Influenza virus B, Influenza virus B-yamagata, Influenza virus C, Influenza virus D, Measles morbillivirus morbillivirus), MERS-CoV, Mumps orthorubulavirus, Nipahvirus, Parainfluenza virus 1, Parainfluenza virus 2, Parainfluenza virus 3, Parainfluenza virus 4a, Parainfluenza virus 4b, Respiratory Syncytial virus A, Respiratory Syncytial virus B, Rhinovirus A, Rhinovirus B, Rhinovirus C,Provided is a composition for simultaneous detection of Rubivirus rubellae, SARS-CoV-2, SARS-CoV, and Varicella zoster virus.

[0013] In addition, the present invention provides a composition for diagnosing respiratory syndrome, including a probe set for simultaneous detection of the respiratory syndrome causative agent.

[0014] In addition, the present invention provides a respiratory syndrome diagnostic kit including the respiratory syndrome diagnostic composition and instructions for use.

[0015] In another example of the present invention, the respiratory syndrome is anthrax, Q fever, tularemia, plague, pertussis, diphtheria, scarlet fever, legionellosis, tuberculosis, Haemophilus influenzae infection, meningococcal meningitis, pneumococcal infection, Mycoplasma pneumoniae infection, Chlamydia pneumoniae infection, psittacosis, blastomycosis, coccidioidomycosis, histoplasmosis, nontuberculous mycobacterial infection, measles, mumps, rubella, adenovirus infection, bocavirus infection, coronavirus infection, severe acute respiratory syndrome, Middle East respiratory syndrome, coronavirus disease-19, metapneumovirus infection, respiratory syncytial virus infection, influenza virus infection, parainfluenza virus infection, rhinovirus infection, chickenpox, Epstein-Barr virus infection, cytomegalovirus infection, herpesvirus 6 It may be at least one selected from the group consisting of infectious diseases, hemorrhagic fever with renal syndrome, enterovirus infection, parechovirus infection, Nipah virus infection, hennipah virus infection, and hendra virus infection.

[0016] The present invention comprises the steps of: preparing an isolated DNA or RNA sample; reacting the isolated DNA or RNA sample using the probe set; and a step of obtaining a detection result, including Bacillus anthracis, Blastomyces dermatitidis, Bordetella pertussis, Chlamydia pneumoniae, Chlamydia psittaci, Coccidioides immitis, Coccidioides posadasii, Corynebacterium diphtheria, Coxiella burnetii, Francisella tularensis, Haemophilus influenzae, Histoplasma capsulatum, Legionella pneumophila, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansassi, Mycobacterium tuberculosis, Mycobacterium abscessus, Mycoplasmoides pneumoniae, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes, Yersinia pestis, Coronavirus 229E, Coronavirus HKU1, Coronavirus NL63,Coronavirus OC43, Cytomegalovirus, Epstein-Barr virus, Hantavirus, Hendravirus, Henipavirus, Human adenovirus type 14, Human adenovirus type 1, Human adenovirus type 21, Human adenovirus type 2, Human adenovirus type 3, Human adenovirus type 4, Human adenovirus type 55, Human adenovirus type 5, Human adenovirus type 7, Human adenovirus Human adenovirus type 8, human adenovirus type, human bocavirus 1, human bocavirus 2, human bocavirus 3, human bocavirus 4, human enterovirus CV-A21, human enterovirus EV-A71, human enterovirus EV-C104, human enterovirus EV-C105, human enterovirus EV-C105, human enterovirus EV-C109,Human enterovirus EV-C117, Human enterovirus EV-C118, Human enterovirus EV-D68, Human herpesvirus 6, Human metapneumovirus A, Human metapneumovirus B, Human parechovirus, Influenza virus A-H1N1, Influenza virus A-H3N2, Influenza virus A, Influenza virus B-victoria, Influenza virus B, Influenza virus B-yamagata (Influenza virus B-yamagata), Influenza virus C, Influenza virus D, Measles morbillivirus, MERS-CoV, Mumps orthorubulavirus, Nipahvirus, Parainfluenza virus 1, Parainfluenza virus 2, Parainfluenza virus 3, Parainfluenza virus 4a, Parainfluenza virus 4b, Respiratory Syncytial virus A,Provided is a method for simultaneous detection of respiratory syncytial virus B, rhinovirus A, rhinovirus B, rhinovirus C, Rubivirus rubellae, SARS-CoV-2, SARS-CoV, and Varicella zoster virus.

[0017] In one example of the present invention, the sample for detection or diagnosis may be isolated from feces, blood, serum, urine, sputum or biological tissue.

[0018] The probe set for simultaneous detection of viruses and bacteria based on respiratory syndrome of the present invention enables simultaneous detection of multiple pathogens through NGS, and secures work efficiency by developing a kit with improved sensitivity, specificity, and accuracy, and can be utilized as a multiple detection system for respiratory syndrome-causing pathogens.

[0019] In addition, it can be actively utilized as a preemptive response before a respiratory syndrome pandemic, and can be expanded to respond to other new infectious diseases that may occur in the future.

[0020] Figure 1 is a schematic diagram of a multi-pathogen panel analysis pipeline according to one embodiment of the present invention.

[0021] Hereinafter, preferred embodiments of the present invention will be described in detail. Furthermore, the following description depicts numerous specific details, such as specific components. However, these are provided to facilitate a more comprehensive understanding of the present invention, and it will be apparent to those skilled in the art that the present invention can be practiced without these specific details. Furthermore, in describing the present invention, if a detailed description of a related, known function or configuration is deemed to unnecessarily obscure the gist of the present invention, such detailed description will be omitted.

[0022]

[0023] <Example 1> Kit component design - separate development of virus and bacterial panels

[0024] Among the pathogens that can cause respiratory syndrome, 62 pathogens were selected for the virus panel and 23 pathogens were selected for the bacteria panel, taking into account the frequency of occurrence and risk.

[0025] Referring to existing testing methods, for the virus panel, the entire genome region was determined as the target region for all pathogens, and for the bacterial panel, specific genes were selected to distinguish pathogens, and the capture region was determined to include the surrounding region to ensure capture performance.

[0026] The pathogen types and reference genome sequence information included in the developed respiratory syndrome virus panel are as shown in Table 1.

[0027] Pathogen Information Panel Target Area Pathogen Name Infectious Disease Name Ref ID Genome Size (bp) Measles morbillivirus Measles AY486083.115,849 Mumps orthorubulavirus Mumps GU980052.115,384 Rubivirus rubellae Rubella FJ211587.19,762 Human adenovirus AF (47 types) Adenovirus OQ518256.134,125 Human adenovirus type 1 (HAdV-C1) Adenovirus OQ518259.135,979 Human adenovirus type 2 (HAdV-C2) Adenovirus OQ518261.135,938 Human adenovirus type 3 (HAdV-B3) Adenovirus OQ518260.135,258 Human Adenovirus type 4 (HAdV-E4) Adenovirus OP785759.135,948 Human adenovirus type 5 (HAdV-C5) Adenovirus OQ518263.135,906 Human adenovirus type 7 (HAdV-B7) Adenovirus OP815345.135,225 Human adenovirus type 8 (HAdV-D8) Adenovirus AB701748.134,997 Human adenovirus type 14 (HAdV-B14) Adenovirus OQ518258.134,754 Human adenovirus type 21 (HAdV-B21) Adenovirus OQ518284.135,371 Human adenovirus type 55 (HAdV-B55) Adenovirus OM714808.134,761 Human bocavirus 1 Human bocavirus OP255990.15,464 Human bocavirus 2 Human bocavirus MG953831.15,141 Human bocavirus 3 Human bocavirus NC_012564.15,242 Human bocavirus 4 Human bocavirus NC_012729.25,104 Coronavirus OC43 Human coronavirus NC_006213.130,741Coronavirus 229E typeHuman coronavirusKY073747.127,837Coronavirus NL63 typeHuman coronavirusNC_005831.227,553Coronavirus HKU1 typeHuman coronavirusNC_006577.229,926SARS-CoV Severe acute respiratory syndromeAY27874129,727MERS-CoV Middle East respiratory syndromeMK12925330,150SARS-CoV-2 COVID-19 virusMN90894729,903Human metapneumovirus type A Human metapneumovirusOL794355.113,376Human metapneumovirus type B Human metapneumovirusOL794356.113,293Respiratory Syncytial virus Respiratory syncytial virus type A OR143134.115,221 Respiratory Syncytial virus type B respiratory syncytial virus OR143225.115,221 Influenza virus type A MH022713.1MH022714.1MH022715.1MH022716.1MH022717.1MH022718.1MH022719.1MH022720.113,571 Influenza virus Influenza virus type A (H1N1pdm09) MG770141.1 MG770142.1 MG770143.1 MG770144.1 MG770145.1 MG770146.1 MG770147.1 MG770148.113,148 Influenza virus type A (H3N2) MF673162.1 MF673163.1 MF673164.1 MF673165.1 MF673166.1 MF673167.1 MF673168.1 MF673169.113,077 Influenza virus Influenza virus type B NC_002204.1NC_002205.1NC_002206.1NC_002207.1NC_002208.1NC_002209.1NC_002210.1NC_002211.114,452 Influenza virus type B (Victoria) influenza virus AF102022.1AF102005.1AF101988.1AF100376.1AF100359.1M58428.111,496Influenza virus Type B (Yamagata) influenza virus L49385.1AF102023.1AF102006.1AF101989.1AY139081.1AF100396.1AF100378.1M58419.113,287Influenza virus Type C influenza virus NC_006306.2NC_006307.2NC_006308.2NC_006309.2NC_006310.2NC_006311.2NC_006312.212,906Influenza virus Influenza virus type D NC_036615.1NC_036616.1NC_036617.1NC_036618.1NC_036619.1NC_036620.1NC_036621.112,800 Parainfluenza virus type 1 Parainfluenza virus NC_003461.115,600 Parainfluenza virus type 2 Parainfluenza virus NC_003443.115,646 Parainfluenza virus type 3 Parainfluenza virus NC_075446.115,462 Parainfluenza virus type 4A Parainfluenza virus NC_021928.117,052 Parainfluenza virus type 4B Parainfluenza virus ON729321.117,219 Human Rhinovirus A Rhinovirus NC_001617.17,152 Human Rhinovirus B Rhinovirus NC_001490.17,212 Human Rhinovirus CRhinovirus NC_009996.17,099 Varicella zoster virus Chickenpox MW545808124,760 Epstein-Barr virus NC_009334.1172,764 Cytomegalovirus NC_006273.2235,646 Human herpesvirus 6 NC_000898.1162,114 Hantavirus Hemorrhagic fever with renal syndrome NC_077666.1 NC_077667.1 NC_077668.112,016 Human Enterovirus CV-A21 Enterovirus infection MZ396299.17,393 Human Enterovirus EV-C104 Enterovirus infection MZ092702.17,390 Human Enterovirus EV-C105 Enterovirus infection MH229997.17,316 Human Enterovirus EV-C109 Enterovirus infection MH128992.17,336 Human Enterovirus EV-C117 Enterovirus infection MT338567.17,363 Human Enterovirus EV-D68 Enterovirus infection NC_038308.17,367 Human Enterovirus EV-A71 Enterovirus infection OQ842403.17,390 Human Enterovirus EV-C118 Enterovirus infection JX678288.17,374 Human parechovirusParechovirus infectionOP255974.17,340NipahvirusNipah virus infectionNC_002728.118,246HenipavirusHenipah virus infectionNC_025351.118,162HendravirusHendra virus infectionNC_001906.318,234.

[0028] The pathogen types and reference genome sequence information included in the developed respiratory syndrome-bacterial panel are as shown in Table 2.

[0029] Pathogen Information Panel Target Area Pathogen Name Infectious Disease Name Ref ID Start End Target Information Bacillus anthracis Anthrax AE017336.2143779147295 pagA ~ pagRBacillus anthracis Anthrax AE017335.35559956993 capBBacillus anthracis Anthrax AE017334.2505920527082 pflA ~ sspE ~ hemL1 Coxiella burnetii Q fever NC_002971.410049281028201 scpB ~ (IS1111) ~ mutLFrancisella tularensis Tularemia AJ749949.2902372924525 purCD ~ lpnA ~ ileSYersinia pestis plague CP002956.137490763770553A1122_17515 ~ (yapJ) ~A1122_17575Yersinia pestis plague CP002958.125333957A1122_21777 ~(pla)~ A1122_21782Yersinia pestis plague CP002957.15655460120A1122_21602 ~ (F1 operon)~ A1122_21612Bordetella pertussis pertussis CP025371.17574997123vir89 ~ ptxA(CXD86_00450) ~ murACorynebaterium diphtheriaNC002935.2395838417308musG ~rpoB ~ rpoCGroup A β-hemollyticStreptococcus(streptococcus_pyogenes)Scarlet feverAE00656510238061046123pstB ~ (spy)~ pcrALegionella spp.LegionellosisAE017354.1154334176453dapB ~ (ssrA)~ ccrBMycobacterium tuberculosistuberculosisNC000962.3519600533789sodC ~ Hsp65(groEL2) ~ rskAMycobacterium tuberculosistuberculosisNC000962.3756137767320atsD ~ rpoB ~rpoCHaemophilus influenzaeHaemophilus influenzae infectionLR134168309064331741tnaA ~ (protein D)~ qlpTNeisseria meningitidisMeningococcal meningitisNZ_CP0215209888801010738CCD84_05780 ~ SodC(CCD84_05830) ~ CCD84_05875Streptococcus pneumoniaePneumococcal infectionNZ_CP02054918356011858697SPNHU17_01976 ~ (lytA)~ rpoBMycoplasma pneumoniaeMycoplasma pneumoniae Infection LR214945.16924777135845S rRNA ~ 16S rRNA ~ tktChlamydophila pneumoniaeNC_005043767506789643CpB0716 ~ ompA(CpB0722) ~ fliNChlamydophila psittaci(Chlamydia psittaci)PsittacosisNC_015470437176459560enr-A ~ (CPSIT_RS01985)~ G50_0437Blastomyces dermatitidisBlastomycosisGCA_000003525.2113603441018S rRNA ~ 28S rRNACoccidioides immitisCoccidioidomycosisGCA_000149335.277757457799928CIMG_10511 ~ (Ci45815)~ CIM_G00232Coccidioides posadasiiCoccidioidomycosisGCA_000151335.147733084797287CPC735_059340 ~(Cp45810)~ CPC735_059390Histoplasma capsulatumHistoplasmosisGCA_000150115.1345526840HCBG_12003 ~ (mtSSU)~ HCBG_12007mycobacterium avium, NTMNontuberculous mycobacterial infectionCP018019.143461334370545BJP76_19970 ~rpoB(BJ76_20000) ~BJP76_20045mycobacterium intracellular, NTM nontuberculous mycobacterial infectionCP085945.111368931161906LK403_05395~rpoB(LK403_05455) ~ LK403_05475mycobacterium kansassi, NTM nontuberculous mycobacterial infectionCP006835.142551544284932MKAN_19425~rpoB(MKAN_19465) ~ MKAN_19530mycobacteroides abscessus, NTM nontuberculous Acid-fast bacterial infectionCP034181.110450331070150EFV83_05480 ~ rpoB(EFV83_05485).

[0030] The sequences of the specific probe sets are as follows.

[0031] SEQ ID NO: 1-58: Bacillus anthracis PagA Probe 1-58

[0032] SEQ ID NO: 59-81: Bacillus anthracis capB Probe 1-23

[0033] SEQ ID NO: 82-433: Bacillus anthracis sspE Probe 1-352

[0034] X SEQ ID NO: 434-766: Blastomyces dermatitidis 18S rRNA Probe 1-333

[0035] Sequence ID 767-1122: Bordetella pertussis ptxA Probe 1-356

[0036] X SEQ ID NO: 1123-1490: Chlamydia pneumoniae ompA Probe 1-368

[0037] Sequence ID 1491-1863: Chlamydia psittaci CPSIT_RS01985 Probe 1-373

[0038] X SEQ ID NO: 1864-2266: Coccidioides immitis Ci45815 Probe 1-403

[0039] X SEQ ID NO: 2267-2665: Coccidioides posadasii Cp45810 Probe 1-399

[0040] SEQ ID NO: 2666-3022: Corynebacterium diphtheria rpoB Probe 1-357

[0041] Sequence number 3023-3409: Coxiella burnetii IS1111 Probe 1-387

[0042] SEQ ID NO: 3410-3778: Francisella tularensis lpnA Probe 1-369

[0043] SEQ ID NO: 3779-4155: Haemophilus influenzae protein D Probe 1-377

[0044] X SEQ ID NO: 4156-4544: Histoplasma capsulatum mtSSU Probe 1-389

[0045] Sequence numbers 4545-4912: Legionella pneumophila ssrA Probe 1-368

[0046] X SEQ ID NO: 4913-5318: Mycobacterium avium rpoB Probe 1-406

[0047] X SEQ ID NO: 5319-5734: Mycobacterium intracellulare rpoB Probe 1-416

[0048] X SEQ ID NO: 5735-6230: Mycobacterium kansassi rpoB Probe 1-496

[0049] SEQ ID NO: 6231-6466: Mycobacterium tuberculosis Hsp65 Probe 1-236

[0050] SEQ ID NO: 6467-6652: Mycobacterium tuberculosis rpoB Probe 1-186

[0051] X SEQ ID NO: 6653-7070: Mycobacteroides abscessus rpoB Probe 1-418

[0052] SEQ ID NO: 7071-7421: Mycoplasmoides pneumoniae 16S rRNA Probe 1-351

[0053] SEQ ID NO: 7422-7784: Neisseria meningitidis SodC Probe 1-363

[0054] SEQ ID NO: 7785-8168: Streptococcus pneumoniae lytA Probe 1-384

[0055] SEQ ID NO: 8169-8539: Streptococcus pyogenes spy Probe 1-371

[0056] SEQ ID NO: 8540-8896: Yersinia pestis yapJ Probe 1-357

[0057] Sequence numbers 8897-8919: Yersinia pestis pla Probe 1-23

[0058] SEQ ID NO: 8920-8978: Yersinia pestis F1 operon Probe 1-59

[0059] Sequence ID 8979-9441: Coronavirus 229E Probe 1-463 (whole genome)

[0060] X Sequence number 9442-9935: Coronavirus HKU1 Probe 1-494 (whole genome)

[0061] Sequence ID 9936-10394: Coronavirus NL63 Probe 1-459 (whole genome)

[0062] Sequence numbers 10395-10906: Coronavirus OC43 Probe 1-512 (whole genome)

[0063] Sequence number 10907-14833: Cytomegalovirus Probe 1-3927 (whole genome)

[0064] X SEQ ID NO: 14834-17628: Epstein-Barr virus Probe 1-2795 (whole genome)

[0065] Sequence numbers 17629-17827: Hantavirus Probe 1-199 (whole genome)

[0066] X SEQ ID NO: 17828-18130: Hendravirus Probe 1-303 (whole genome)

[0067] X Sequence number 18131-18432: Henipavirus Probe 1-302 (whole genome)

[0068] X Sequence number 18433-19011: Human adenovirus type 14 Probe 1-579 (whole genome)

[0069] Sequence number 19012-19610: Human adenovirus type 1 Probe 1-599 (whole genome)

[0070] X Sequence number 19611-20199: Human adenovirus type 21 Probe 1-589 (whole genome)

[0071] Sequence number 20200-20797: Human adenovirus type 2 Probe 1-598 (whole genome)

[0072] Sequence number 20798-21384: Human adenovirus type 3 Probe 1-587 (whole genome)

[0073] Sequence numbers 21385-21983: Human adenovirus type 4 Probe 1-599 (whole genome)

[0074] Sequence number 21984-22562: Human adenovirus type 55 Probe 1-579 (whole genome)

[0075] Sequence number 22563-23160: Human adenovirus type 5 Probe 1-598 (whole genome)

[0076] Sequence number 23161-23747: Human adenovirus type 7 Probe 1-587 (whole genome)

[0077] Sequence number 23748-24330: Human adenovirus type 8 Probe 1-583 (whole genome)

[0078] Sequence number 24331-24898: Human adenovirus Probe 1-568 (whole genome)

[0079] Sequence number 24899-24989: Human bocavirus 1 Probe 1-91 (whole genome)

[0080] X Sequence number 24990-25074: Human bocavirus 2 Probe 1-85 (whole genome)

[0081] X Sequence number 25075-25161: Human bocavirus 3 Probe 1-87 (whole genome)

[0082] X Sequence number 25162-25246: Human bocavirus 4 Probe 1-85 (whole genome)

[0083] X Sequence number 25247-25369: Human enterovirus CV-A21 Probe 1-123 (whole genome)

[0084] Sequence numbers 25370-25492: Human enterovirus EV-A71 Probe 1-123 (whole genome)

[0085] X Sequence number 25493-25615: Human enterovirus EV-C104 Probe 1-123 (whole genome)

[0086] X Sequence number 25616-25736: Human enterovirus EV-C105 Probe 1-121 (whole genome)

[0087] X Sequence number 25737-25858: Human enterovirus EV-C109 Probe 1-122 (whole genome)

[0088] X Sequence number 25859-25980: Human enterovirus EV-C117 Probe 1-122 (whole genome)

[0089] X Sequence number 25981-26102: Human enterovirus EV-C118 Probe 1-122 (whole genome)

[0090] Sequence numbers 26103-26224: Human enterovirus EV-D68 Probe 1-122 (whole genome)

[0091] X Sequence number 26225-28915: Human herpesvirus 6 Probe 1-2691 (whole genome)

[0092] X SEQ ID NO: 28916-29137: Human metapneumovirus A Probe 1-222 (whole genome)

[0093] SEQ ID NO: 29138-29358: Human metapneumovirus B Probe 1-221 (whole genome)

[0094] Sequence numbers 29359-29480: Human parechovirus Probe 1-122 (whole genome)

[0095] Sequence numbers 29481-29695: Influenza virus A-H1N1 Probe 1-215 (whole genome)

[0096] Sequence numbers 29696-29909: Influenza virus A-H3N2 Probe 1-214 (whole genome)

[0097] X Sequence number 29910-30131: Influenza virus A Probe 1-222 (whole genome)

[0098] Sequence numbers 30132-30320: Influenza virus B-victoria Probe 1-189 (whole genome)

[0099] Sequence numbers 30321-30556: Influenza virus B Probe 1-236 (whole genome)

[0100] Sequence numbers 30557-30774: Influenza virus B-yamagata Probe 1-218 (whole genome)

[0101] X Sequence number 30775-30986: Influenza virus C Probe 1-212 (whole genome)

[0102] X Sequence number 30987-31196: Influenza virus D Probe 1-210 (whole genome)

[0103] Sequence numbers 31197-31460: Measles morbillivirus Probe 1-264 (whole genome)

[0104] Sequence numbers 31461-31962: MERS-CoV Probe 1-502 (whole genome)

[0105] Sequence numbers 31963-32218: Mumps orthorubulavirus Probe 1-256 (whole genome)

[0106] X Sequence number 32219-32522: Nipahvirus Probe 1-304 (whole genome)

[0107] Sequence number 32523-32781: Parainfluenza virus 1 Probe 1-259 (whole genome)

[0108] Sequence number 32782-33041: Parainfluenza virus 2 Probe 1-260 (whole genome)

[0109] Sequence number 33042-33298: Parainfluenza virus 3 Probe 1-257 (whole genome)

[0110] Sequence numbers 33299-33582: Parainfluenza virus 4a Probe 1-284 (whole genome)

[0111] Sequence numbers 33583-33868: Parainfluenza virus 4b Probe 1-286 (whole genome)

[0112] Sequence number 33869-34121: Respiratory Syncytial virus A Probe 1-253 (whole genome)

[0113] Sequence numbers 34122-34374: Respiratory Syncytial virus B Probe 1-253 (whole genome)

[0114] Sequence numbers 34375-34493: Rhinovirus A Probe 1-119 (whole genome)

[0115] Sequence numbers 34494-34614: Rhinovirus B Probe 1-121 (whole genome)

[0116] Sequence numbers 34615-34732: Rhinovirus C Probe 1-118 (whole genome)

[0117] Sequence numbers 34733-34894: Rubivirus rubellae Probe 1-162 (whole genome)

[0118] Sequence numbers 34895-35392: SARS-CoV-2 Probe 1-498 (whole genome)

[0119] Sequence numbers 35393-35887: SARS-CoV Probe 1-495 (whole genome)

[0120] *Sequence number 35888-37966: Varicella zoster virus Probe 1-2079 (whole genome)

[0121] A prototype kit containing all reagent components required for the entire experimental process from nucleic acid samples isolated from specimens to NGS analysis equipment was produced.

[0122] - Basic production components: NGS Library production module, Target Capture module, Respiratory Syndrome Pathogen Capture Panel (consisting of virus panel and bacteria panel respectively).

[0123] - Additional components provided: cDNA synthesis module, polymerase, magnetic beads, streptavidin beads.

[0124] The detailed time required for each experimental step of the developed kit is as shown in Table 3 below.

[0125] Detailed stepsTime requiredcDNASynthesis1st & 2nd strand cDNA synthesis2 hrPurification (clean-up)40 minNGS LibraryPreparationEnzymatic preparation1 hrAdapter ligation20 minPurification (clean-up)40 minAmplification20 minPurification (clean-up)40 minQC30 minHybridizationPrepare reagents for Hybridization1 hrTarget capture16 hr(overnight)Selection of target captured library1.5 hrAmplification & QC1.5 hrTotal time required26 hr 10 min

[0126] <Example 2> Kit component design - NGS analysis pipeline development

[0127] We developed a dedicated pipeline capable of analyzing NGS data produced using a respiratory syndrome multi-pathogen detection panel.

[0128] Since the real specimen contains nucleic acids derived from the human genome as well as the pathogen genome, a two-step analysis method was applied to first confirm human genome-derived data from the NGS raw data and then confirm pathogen information from the remaining data after excluding this (see Figure 1).

[0129] The developed analysis pipeline can be packaged as a Docker container, allowing it to be easily installed and used in various analysis environments without preparing individual programs and libraries.

[0130] Analysis results are provided in visualized reports in PDF and CSV formats, including NGS data quality, the amount of pathogen data, and whether pathogens were detected. In addition, mutation information, depth information by location, and read mapping information for observed pathogens can be provided.

[0131] <Example 3> Performance Verification - In Silico Analysis Using Simulation Data

[0132] We developed an in silico analysis program that generates simulation data based on reference genome sequences for pathogens included in the panel and can confirm whether each pathogen can be detected.

[0133] In silico analysis using simulation data can estimate the performance of kits for pathogens for which real samples are difficult to obtain, and can predict how the kit will respond when new mutations occur in each pathogen.

[0134] Detection of each pathogen is based on 10x coverage (20%), which means that NGS reads for the target gene region of the pathogen are observed an average of 10 times or more in 20% or more of the entire target region (Table 2).

[0135] - The 10x coverage, which is the pathogen detection standard, was established through preliminary investigation of commercially available pathogen detection products (Company I, Company T, etc.).

[0136] Coverage Range Detection Decision 10x Cov ≥ 20% Detected 10% ≤ 10x Cov < 20% Indeterminate 10x Cov < 10% Not Detected

[0137] Using the developed in silico analysis program, simulation NGS data was produced and analyzed assuming that mutations occurred at a frequency of 10% in the reference genome sequence for the target genes of each pathogen. The results are shown in Table 5-6.

[0138] 샘플명검출 병원체명10x Coverage(%)Coronavirus_229E_refCoronavirus 229E98.30Coronavirus_HKU1_refCoronavirus HKU198.44Coronavirus_NL63_refCoronavirus NL6398.80Coronavirus_OC43_refCoronavirus OC4398.65Human_metapneumovirus_A_refHuman metapneumovirus A97.33Human_metapneumovirus_B_refHuman metapneumovirus B97.10MERS-CoV_refMERS-CoV98.24Parainfluenza_virus_1_refParainfluenza virus 198.10Parainfluenza_virus_2_refParainfluenza virus 298.16Parainfluenza_virus_3_refParainfluenza virus 398.10Parainfluenza_virus_4A_refParainfluenza virus 4A97.37Parainfluenza_virus_4B_refParainfluenza virus 4B97.74Respiratory_Syncytial_virus_A_refRespiratory Syncytial virus A98.25Respiratory_Syncytial_virus_B_refRespiratory Syncytial virus B97.92Rhinovirus_A_refRhinovirus A97.27Rhinovirus_B_refRhinovirus B97.27Rhinovirus_C_refRhinovirus C96.96SARS-CoV-2_refSARS-CoV-297.49SARS-CoV_Urbani_refSARS-CoV98.40cytomegalovirus_refCytomegalovirus98.28epstein-barr_virus_refEpstein-Barr virus98.43hantavirus_refHantavirus96.92hendravirus_refHendravirus98.21henipavirus_refHenipavirus98.38human_adenovirus_refHuman adenovirus98.56human_adenovirus_type14_refHuman adenovirus type 1497.91human_adenovirus_type1_refHuman adenovirus type 198.34human_adenovirus_type21_refHuman adenovirus type 2198.38human_adenovirus_type2_refHuman adenovirus type 297.94human_adenovirus_type3_refHuman adenovirus type 398.22human_adenovirus_type4_refHuman adenovirus type 498.36human_adenovirus_type55_refHuman adenovirus type 5598.38human_adenovirus_type5_refHuman adenovirus type 598.25human_adenovirus_type7_refHuman adenovirus type 797.20human_adenovirus_type8_refHuman adenovirus type 896.93human_bocavirus1_refHuman bocavirus 196.85human_bocavirus2_refHuman bocavirus 296.30human_bocavirus3_refHuman bocavirus 397.80human_bocavirus4_refHuman bocavirus 496.90human_enterovirus_CV-A21_refHuman enterovirus CV-A2197.80human_enterovirus_EV-A71_refHuman enterovirus EV-A7195.91human_enterovirus_EV-C104_refHuman enterovirus EV-C10495.82human_enterovirus_EV-C105_refHuman enterovirus EV-C10594.57human_enterovirus_EV-C109_refHuman enterovirus EV-C10995.38human_enterovirus_EV-C117_refHuman enterovirus EV-C11796.51human_enterovirus_EV-C118_refHuman enterovirus EV-C11895.93human_enterovirus_EV-D68_refHuman enterovirus EV-D6896.23human_herpesvirus_6_refHuman herpesvirus 698.40human_parechovirus_refHuman parechovirus98.70influenza_A_virus-H1N1_refInfluenza A virus-H1N194.43influenza_A_virus-H3N2_refInfluenza A virus-H3N294.60influenza_A_virus_refInfluenza virus93.46influenza_B_virus-victoria_refInfluenza B virus-victoria94.30influenza_B_virus-yamagata_refInfluenza B virus-yamagata93.97influenza_B_virus_refInfluenza B virus95.70influenza_C_virus_refInfluenza C virus94.96Influenza_D_virus_D virus95.58measles_morbillivirus_refMeasles morbillivirus98.19mump_orthorubulavirus_refMump orthorubulavirus98.26nipahvirus_refNipahvirus98.18rubivirus_rubellae_refRubivirus rubellae97.35varicella_zoster_virus_refVaricella zoster virus98.76.

[0139] 샘플명검출 병원체명10x Coverage(%)bacillus_anthracis.targetBacillus anthracis97.99blastomyces_dermatitidis.targetBlastomyces dermatitidis99.62bordetella_pertussis.targetBordetella pertussis97.98chlamydia_pneumoniae.targetChlamydia pneumoniae98.10chlamydia_psittaci.targetChlamydia psittaci98.14coccidioides_immitis.targetCoccidioides immitis98.90coccidioides_posadasii.targetCoccidioides posadasii97.87corynebacterium_diphtheriae.targetCorynebacterium diphtheriae98.31coxiella_burnetii.targetCoxiella burnetii95.93francisella_tularensis.targetFrancisella tularensis98.28haemophilus_influenzae.targetHaemophilus influenzae98.42histoplasma_capsulatum.targetHistoplasma capsulatum98.24legionella_pneumophila.targetLegionella pneumophila97.76mycobacterium_avium-NTM.targetMycobacterium avium-NTM98.42mycobacterium_intracellulare-NTM.targetMycobacterium intracellulare-NTM98.70mycobacterium_kansassi-NTM.targetMycobacterium kansassi-NTM98.16mycobacterium_tuberculosis.targetMycobacterium tuberculosis98.38mycobacteroides_abscessus-NTM.targetMycobacteroides abscessus-NTM97.88mycoplasmoides_pneumoniae.targetMycoplasmoides pneumoniae98.00neisseria_meningitidis.targetNeisseria meningitidis97.80streptococcus_pneumoniae.targetStreptococcus pneumoniae98.16streptococcus_pyogenes.targetStreptococcus pyogenes98.28yersinia_pestis.targetYersinia pestis94.84.

[0140] In silico analysis revealed no cross-reactivity between reference genomes within the respiratory syndrome-virus panel and the respiratory syndrome-bacterial panel.

[0141] To predict the capture performance according to strain / subtype, the produced NGS simulation data was applied to various strains / subtypes and a reactivity analysis was conducted.

[0142] Most of the 62 strains included in the virus panel had 10x coverage greater than 70% at the strain level.

[0143] Reference NameStrain IDTaget size (bp)10x Coverage(%)Coronavirus_HKU1_refON553962.13014476.33Coronavirus_HKU1_refOK073085.12995397.74Coronavirus_HKU1_refON128609.12969597.81Coronavirus_HKU1_refMK167038.12992178.81Coronavirus_HKU1_refMH940245.12981177.88Coronavirus_HKU1_refLC315651.22990377.72cytomegalovirus_refOU342900.123618089.27cytomegalovirus_refMW528462.123469790.12cytomegalovirus_refKY490085.123563289.34cytomegalovirus_refKY123652.123523390.53cytomegalovirus_refKY002201.123568189.75cytomegalovirus_refKX544841.122230991.05hantavirus_refNC_077666.1, NC_077667.1, NC_077668.11201696.92hantavirus_refNC_005223.1, NC_005224.1, NC_005225.1120626.50hantavirus_refNC_003466.1, NC_003467.2, NC_003468.2121041.37hantavirus_refKT885050.1, KT885051.1, KT885052.11201696.83hantavirus_refON661335.1, ON661337.1, OP094683.1118440.35hantavirus_refKX079474.1, KX079475.1, KX079476.1119560.00hantavirus_refOM030305.1, OM030306.1, OM030307.1118450.00hendravirus_refMZ229746.11823418.81hendravirus_refMZ229748.11823418.52hendravirus_refJN255806.11823496.96hendravirus_refHM044321.11823497.72hendravirus_ref AF017149.31823498.21henipavirus_refNC_025352.1184060.00henipavirus_refNC_025256.1185300.00henipavirus_refOM101125.1184020. 00henipavirus_refOK623353.1199440.00henipavirus_refOK623354.1197460.00Rhinovirus_A_refFJ445170.1711000.00Rhinovirus_A_refM Z670593.1708413.89Rhinovirus_A_refM16248.1715297.27Rhinovirus_A_refOL770292.1713300.00Rhinovirus_A_refFJ445166.1715253.97.

[0144] Some species included in the virus panel (hantavirus, hendravirus, henipavirus, rhinovirus, adenovirus, enterovirus, etc.) had very low genomic similarity between strains / subtypes, so 10x coverage values ​​were not calculated or very low coverage values ​​were observed.

[0145] - Analysis was conducted on approximately 1,700 genomes among the strain / subtype genome data collected for 62 virus species (Table 7 shows only a portion of the results).

[0146] It was confirmed that for strains with sequence similarity of less than approximately 80% with the reference genome, low coverage values ​​were obtained because the mapping of NGS simulation reads generated based on the reference genome was not accurate.

[0147] In order to determine how much of a difference there is between the reference genome and the strains, we conducted a genome similarity analysis between strains for species with very low 10x coverage values, such as Henipavirus and Rhinovirus A (Table 8-10).

[0148] For the Coronavirus HKU1 strain, the strain similarity was observed to be over 90%, which may be information that supports the good 10x coverage value in Table 7.

[0149] For Henipavirus and Rhinovirus A, the similarity between strains was observed to be between 56% and 100%, and when compared to Table 7, it can be confirmed that the 10x coverage value was low or calculated as 0 when the genome similarity was less than 80%.

[0150] Coronavirus_HKU1NC_006577.2ON553962.1OK73085.1ON128609.1MK167038.1MH940245.1LC315651.2NC_006577.2-ON553962.190.37-OK073085.199.0890.13 -ON128609.198.2789.6598.33-MK167038.191.6197.7791.3090.78-MH940245. 191.0598.1090.7990.7998.36-LC315651.291.1398.8990.8990.4698.6599.07-

[0151] HenipavirusNC_025351.1NC_025352.1NC_025256.1OM101125.1OK623353.1OK623354.1NC_025351.1-NC_025352.156.57-NC_025 256.159.1459.44-OM101125.156.3570.4559.05-OK623353.156.6363.7658.8163.92-OK623354.157.1563.4159.3864.1268.27-

[0152] Rhinovirus ANC_001617.1FJ445170.1MZ670593.1M16248.1OL770292.1FJ445166.1NC_001617.1-FJ445170.159.92-MZ670593.176 .1660.13-M16248.1100.0059.9276.16-OL770292.163.7161.5563.0763.71-FJ445166.185.3559.8775.2685.3563.54-

[0153] Even in cases where the genomic similarity between strains is low, such as Henipavirus and Rhinovirus A in Table 8-10, it is expected that the capture performance for other strains can be maintained through the high mutation response of the probe sequences used in the target capture panel, and it is thought that the detection of target species will be possible through the newly developed de novo assembly-based analysis pipeline.

[0154] When the same analysis was performed on various strains of pathogens included in the bacterial panel, a 10x coverage of 68% to 100% was observed (Table 11).

[0155] - Analysis was conducted on approximately 300 strain genome data collected for 23 bacterial species (Table 11 shows only a portion of the results).

[0156] Reference NameStrain IDTarget size(bp)10x Coverage(%)Bacillus anthracisGCA_000007845.1_ASM784v1_genomic2607580.03Bacillus anthracisGCA_000008165.1_ASM816v1_genomic2607580.03Bacillus anthracisGCA_000008445.1_ASM844v1_genomic2607598.06Bacillus anthracisGCA_000021445.1_ASM2144v1_genomic2607597.99Bacillus anthracisGCA_000022865.1_ASM2286v1_genomic2607598.06Bacillus anthracisGCA_000258885.1_ASM25888v1_genomic2607598.01Bacillus anthracisGCA_000295695.2_ASM29569v2_genomic2607598.01Bacillus anthracisGCA_022221085.1_ASM2222108v1_genomic2607598.03Bordetella pertussisGCA_013168315.1_ASM1316831v1_genomic2137597.98Bordetella pertussisGCA_013168595.1_ASM1316859v1_genomic2137598.00Bordetella pertussisGCA_013168615.1_ASM1316861v1_genomic2137598.00Bordetella pertussisGCA_013168635.1_ASM1316863v1_genomic2137598.00Bordetella pertussisGCA_013168655.1_ASM1316865v1_genomic2137598.00Bordetella pertussisGCA_013168675.1_ASM1316867v1_genomic2137597.98Bordetella pertussisGCA_013168695.1_ASM1316869v1_genomic2137598.69Bordetella pertussisGCA_013168715.1_ASM1316871v1_genomic2137598.21Bordetella pertussisGCA_004008975.1_ASM400897v1_genomic2137597.98Coccidioides posadasiiGCA_000151335.1_JCVI-cpa1-1.0_genomic2398097.87Coccidioides posadasiiGCA_000150055.1_ASM15005v1_genomic2398097.28Coccidioides posadasiiGCA_000150185.1_ASM15018v1_genomic2398096.76Coccidioides posadasiiGCA_000150215.1_ASM15021v1_genomic2398091.36Coccidioides posadasiiGCA_000150245.1_ASM15024v1_genomic2398081.18Coccidioides posadasiiGCA_000150555.1_ASM15055v1_genomic2398073.99Coccidioides posadasiiGCA_000150585.1_ASM15058v1_genomic2398074.03Coccidioides posadasiiGCA_000150615.1_ASM15061v1_genomic2398077.52Coccidioides posadasiiGCA_018416015.2_ASM1841601v2_genomic2398097.52Haemophilus influenzaeGCA_000012185.1_ASM1218v1_genomic2267884.55Haemophilus influenzaeGCA_000016465.1_ASM1646v1_genomic2267879.96Haemophilus influenzaeGCA_000931605.1_ASM93160v1_genomic2267886.21Haemophilus influenzaeGCA_000931625.1_ASM93162v1_genomic2267885.18Haemophilus influenzaeGCA_000968335.1_ASM96833v1_genomic2267885.84Haemophilus influenzaeGCA_003425955.1_ASM342595v1_genomic2267884.50Haemophilus influenzaeGCA_008586745.1_ASM858674v1_genomic2267868.57Haemophilus influenzaeGCA_028535035.1_ASM2853503v1_genomic2267879.90Haemophilus influenzaeGCA_919949215.1_KRLund_NTHi_Assembly_Genome3655_genomic2267886.05Haemophilus influenzaeGCA_900635795.1_33962_B02_genomic2267899.85.

[0157] <Example 4> Performance Verification - Respiratory Syndrome-Virus Panel Validation

[0158] Of the 62 pathogens to be detected by the respiratory syndrome virus panel, 35 were provided by the Korea Disease Control and Prevention Agency (KDCA), and the panel validity was evaluated for a total of 40 pathogens, including 5 pathogens purchased in-house.

[0159] It was determined that 22 pathogens for which samples could not be obtained could be detected based on in silico results, and the information on the pathogens detected through the validity test analysis (2 repetitions) using a total of 40 samples was as shown in Table 12.

[0160] 사전명함로1차이스2차스트검출 피이체10x coverage (%)검출 피이체10x coverage (%)Measles morbillivirusCt 20.22Measles morbillivirus99.91Measles morbillivirus99.91Mumps orthorubulavirusCt 16.68Mumps orthorubulavirus99.92Mumps orthorubulavirus99.88Rubivirus rubellaeCt 22.65Rubivirus rubellae99.53Rubivirus rubellae99.43Human_adenovirus type 1 (HAdV-C1) ** Ct 19.4Human adenovirus99.45Human adenovirus99.92Human_adenovirus type 2 (HAdV-C2) ** Ct 20.218Human adenovirus99.99Human adenovirus99.99Humanadenovirus type 3 (HAdV-B3)Ct 19Human adenovirus type 3100.00Human adenovirus type 3100.00Humanadenovirus type 4 (HAdV-E4)Ct 18.87Human adenovirus type 4100.00Human adenovirus type 4100.00Human adenovirus type 5 (HAdV-C5) ** Ct 20.83Human adenovirus99.98Human adenovirus99.98Human adenovirus type 6 (HAdV-C6)Ct 19.3Human adenovirus98.77Human adenovirus99.43Human adenovirus type 7 (HAdV-B7) *149.2 ng / ulHuman adenovirus type 7100.00Human adenovirus type 7100.00Human adenovirus type 8 (HAdV-D8)Ct 23.9Human adenovirus type 899.99Human adenovirus type 8100.00Human adenovirus type 11 (HAdV-B11)Ct 19.8Human adenovirus100.00Human adenovirus100.00Human adenovirus type 55 (HAdV-B55)Ct 23.4Human adenovirus type 5599.92Human adenovirus type 5599.90Human bocavirusCt 18.5Human bocavirus 199.98Human bocavirus 199.98Coronavirus 229E형Ct 24.7Coronavirus 229E84.07Coronavirus 229E84.42Coronavirus NL63형Ct 22.4Coronavirus NL6399.97Coronavirus NL6399.96Coronavirus OC43형 ※Ct 20Human adenovirus type 399.56Human adenovirus type 399.75SARS-CoVCt 25.5SARS-CoV99.76SARS-CoV99.86MERS-CoVCt 26.4MERS-CoV96.23MERS-CoV99.66SARS-CoV-2Ct 20.1SARS-CoV-299.59SARS-CoV-299.59Human metapneumovirusCt 20.7Human metapneumovirus B99.72Human metapneumovirus B99.75Respiratory syncytial virus A형Ct 21.5Respiratory syncytial virus A99.86Respiratory syncytial virus A99.78Respiratory Syncytial Virus B형Ct 18.5Respiratory Syncytial Virus B99.86Respiratory Syncytial Virus B99.84Influenza Virus A형(H1N1pdm09)Ct 16.7Influenza Virus A-H1N187.66Influenza Virus A-H1N187.95Influenza virus A형(H3N2)Ct 18.5Influenza virus A-H3N299.42Influenza virus A-H3N299.32Influenza virus B형 * 162.1 ng / ulInfluenza virus B99.93Influenza virus B99.93Influenza virus B형(Victoria)Ct 15.3Influenza virus B-victoria86.40Influenza virus B-victoria88.51Influenza virus B형(Yamagata) **Ct 14.5Influenza virus B99.52Influenza virus B99.61Parainfluenza virus 1형Ct 25.19Parainfluenza virus 199.78Parainfluenza virus 199.78Parainfluenza virus 2형Ct 23.82Parainfluenza virus 299.93Parainfluenza virus 299.91Parainfluenza virus 3형Ct 21.58Parainfluenza virus 399.70Parainfluenza virus 399.75Parainfluenza virus 4A형 * 107.9 ng / ulParainfluenza virus 4a99.68Parainfluenza virus 4a99.66Parainfluenza virus 4B형 * 100.1 ng / ulParainfluenza virus 4b99.83Parainfluenza virus 4b99.79Human Rhinovirus ※ Ct 17.2Influenza virus A-H3N288.26Influenza virus A-H3N293.53Varicella zoster virusCt 18.31Varicella zoster virus99.90Varicella zoster virus99.94Cytomegalovirus * 146.6 ng / ulCytomegalovirus94.68Cytomegalovirus97.72Hantavirus *** Ct 20Hantavirus18.47Hantavirus22.01Human Enterovirus EV-A711.8x10 4 copy / ulHuman enterovirus EV-A7179.13Human enterovirus EV-A7181.72Human Enterovirus EV-D681.4x10 4copy / ulHuman enterovirus EV-D6899.99Human enterovirus EV-D6899.92Human parechovirus1.3x10 4 copy / ulHuman parechovirus50.82Human parechovirus50.89

[0161] * pathogens purchased by the company

[0162] ** Pathogens that cannot be classified into subtypes

[0163] *** Pathogens classified as gray zone

[0164] ※ If other pathogens are detected

[0165]

[0166] All 40 virus samples met the detection criteria of 10x coverage (20%) or higher.

[0167] In addition, it was confirmed that Coronavirus OC43 type and Human Rhinovirus were detected as incorrect pathogens as Human adenovirus type 3 and Influenza virus A-H3N2, respectively, and Whole genome sequencing was performed using samples of the two pathogens to analyze the cause (Table 13).

[0168] Sample name Total number of sequencing reads Data multiples of genome Pathogen data ratio (%) Detected pathogen 10x Coverage (%) Coronavirus OC43 type (WGS) 2,674,458 26,273.9 10.85--Coronavirus OC43 * 3,038,60029,851.2572.35Coronavirus OC4399.92Human Rhinovirus (WGS)3,463,014146,229.05870.00--Human Rhinovirus A *2,987,402126,481.901624.72Rhinovirus A33.53

[0169] * Results of respiratory syndrome virus panel test on pathogen samples owned by the company (Coronavirus OC43: Twist Bioscience (103013), Human Rhinovirus A: ATCC (VR-1645))

[0170]

[0171] Coronavirus OC43 and Human Rhinovirus were confirmed to be pathogens in our company, and there was no problem in detection, but there was a difference in the pathogen data ratio, so it seems that the sample quality was affected.

[0172]

[0173] <Example 5> Performance Verification - Respiratory Syndrome-Bacterial Panel Validation

[0174] Among the 23 pathogens detected by the respiratory syndrome virus panel, 14 were provided by the Korea Disease Control and Prevention Agency and the panel validity was evaluated.

[0175] Nine pathogens for which samples could not be obtained were judged to be detectable based on in silico results.

[0176] The pathogen information detected through the validity test analysis using a total of 14 types of samples is as shown in Table 14.

[0177] Sample name Content 1st evaluation 2nd evaluation Detected pathogen 10x coverage (%) Detected pathogen 10x coverage (%) Bacillus anthracis 1x10 6 copy / ulBacillus anthracis99.99Bacillus anthracis99.99Coxiella burnetii1.7x10 3 copy / ul (Ct 25 or less)Coxiella burnetii100.00Coxiella burnetii100.00Francisella tularensis1X106 copy / ulFrancisella tularensis99.68Francisella tularensis99.74Yersinia pestis1X10 6 copy / ulYersinia pestis99.99Yersinia pestis99.99Bordetella pertussisCt 23Bordetella pertussis100.00Bordetella pertussis100.00Corynebaterium diphtheria100ulCorynebacterium diphtheriae95.26Corynebacterium diphtheriae95.26Group A β-hemollyticStreptococcus1X10 5 copy / ul, 100ulStreptococcus pyogenes99.99Streptococcus pyogenes100.00Legionella spp.Ct 25이하Legionella pneumophila100.00Legionella pneumophila100.00Mycobacterium tuberculosis100ulMycobacterium tuberculosis99.99Mycobacterium tuberculosis99.99Haemophilus influenzae2.5x10 6 copy / ulHaemophilus influenzae99.98Haemophilus influenzae99.99Neisseria meningitidis2.5x10 6 copy / ulNeisseria meningitidis98.57Neisseria meningitidis98.71Streptococcus pneumoniae3x10 7copy / ulStreptococcus pneumoniae98.87Streptococcus pneumoniae98.87Mycoplasma pneumoniae100ulMycoplasmoides pneumoniae100.00Mycoplasmoides pneumoniae100.00Chlamydophila psittaci1.2x10 4 copy / ulChlamydia psittaci100.00Chlamydia psittaci100.00

[0178] All 14 pathogens were confirmed to have 10x coverage, or 20% or more.

[0179] <Example 6> Performance Verification - Overall Results of Efficacy Evaluation for Respiratory Syndrome Viruses and Bacterial Pathogens

[0180] The developed capture probe panel for a total of 85 target pathogens was confirmed to detect all of them through in silico analysis and validation tests.

[0181] * 85 types: 62 viruses (22 in silico analyses and 40 efficacy assessments), 23 bacteria (9 in silico analyses and 14 efficacy assessments)

[0182] <Example 7> Performance Verification - Respiratory Syndrome-Virus Panel LoD Evaluation

[0183] Using the evaluation sample whose pathogen content was confirmed, sequentially diluted samples were prepared, and the minimum detectable unit of pathogen was confirmed using the panel.

[0184] The results of the analysis using the respiratory syndrome-virus panel after diluting the samples with confirmed pathogen content are shown in Table 15.

[0185] 샘플명함량1차 평가2차 평가검출 병원체10x coverage (%)검출 병원체10x coverage (%)Mumps orthorubulavirusCt 25Mumps orthorubulavirus99.91Mumps orthorubulavirus99.90Ct 28Mumps orthorubulavirus99.75Mumps orthorubulavirus99.63Ct 31Mumps orthorubulavirus75.02Mumps orthorubulavirus71.33Ct 35----Human adenovirus type 4 (HAdV-E4)Ct 25Human adenovirus type 4100.00Human adenovirus type 4100.00Ct 28Human adenovirus type 4100.00Human adenovirus type 4100.00Ct 32Human adenovirus type 499.98Human adenovirus type 499.97Ct 35Human adenovirus type 488.90Human adenovirus type 488.58SARS-CoV 2Ct 25SARS-CoV-299.83SARS-CoV-299.77Ct 28SARS-CoV-299.58SARS-CoV-299.58Ct 32----Ct 35----SARS-CoV 2 ※ 1x10 3 copiesSARS-CoV-298.83SARS-CoV-299.401x10 2copies--SARS-CoV-287.65Respiratory Syncytial virus B형Ct 25.5Respiratory Syncytial virus B99.88Respiratory Syncytial virus B99.89Ct 29Respiratory Syncytial virus B99.84Respiratory Syncytial virus B99.80Ct 32Respiratory Syncytial virus B99.34Respiratory Syncytial virus B99.38Ct 35Respiratory Syncytial virus B38.44Respiratory Syncytial virus B30.96Influenza virus A형(H3N2)Ct 25.5Influenza virus A-H3N299.46Influenza virus A-H3N295.82Ct 29Influenza virus A-H3N298.70Influenza virus A-H3N298.23Ct 32Influenza virus A-H3N251.96Influenza virus A-H3N246.18Ct 35Influenza virus A-H3N26.16Influenza virus A-H3N28.84Parainfluenza virus 2형Ct 25Parainfluenza virus 299.92Parainfluenza virus 299.92Ct 28Parainfluenza virus 299.92Parainfluenza virus 299.97Ct 31Parainfluenza virus 299.91Parainfluenza virus 299.91Ct 35Parainfluenza virus 299.80Parainfluenza virus 299.80

[0186] ※ 자사 보유 검체 (SARS-CoV-2: 105204, Control 48 (B.1.1.529 / BA.1))

[0187] The LoD was confirmed at Ct 31 for Mumps orthorubulavirus, Ct 28 for SARS-CoV-2, and Ct 32 for Influenza virus type A (H3N2).

[0188] Human adenovirus type 4 (HAdV-E4), Respiratory Syncytial virus type B, and Parainfluenza virus type 2 had a 10x coverage rate of 80% or higher even in 1 / 1000 diluted samples, so the LoD was estimated to be Ct 35 or higher.

[0189] For SARS-CoV-2, we dilute our own samples to 1x10 3 The results of two replicate tests with diluted samples of the same copy content were 10x coverage, 98.83%, and 98.09%, respectively.

[0190] Minimum detection limit (LOD) evaluation results, Ct 28~35, 1x10 3 The sensitivity was confirmed to be good as it was confirmed as copies. However, the results for some pathogens may vary depending on the pathogen.

[0191] In addition to the detected pathogens matching the actual specimen pathogens, human adenovirus types 4 and 7 and parainfluenza virus 4A and 4B were observed to be detected simultaneously in the analysis results of LoD experimental specimens, and the proportion thereof relatively increased as the dilution factor increased, suggesting that this was due to aerosol contamination.

[0192] <Example 8> Performance Verification - Respiratory Syndrome-Bacterial Panel LoD Evaluation

[0193] The results of the analysis using the respiratory syndrome-bacterial panel after diluting the samples with confirmed pathogen content are shown in Table 16.

[0194] Sample name Content 1st evaluation 2nd evaluation Detected pathogen 10x coverage (%) Detected pathogen 10x coverage (%) Bordetella pertussis Ct 25 Bordetella pertussis 100.00 Bordetella pertussis 100.00 Ct 28 Bordetella pertussis 100.00 Bordetella pertussis 99.87 Ct 31 Bordetella pertussis 99.77 Bordetella pertussis 99.60 Ct 34 Bordetella pertussis 99.17 Bordetella pertussis 94.25 Francisella tularensis 1x10 5 copiesFrancisella tularensis99.68Francisella tularensis99.681x10 4 copiesFrancisella tularensis99.18Francisella tularensis99.681x10 3 copies----1x10 2 copies----

[0195] Bordetella pertussis was observed to have a 10x coverage of over 92% even at Ct 34, and therefore the LoD is estimated to be above Ct 34.

[0196] Francisella tularensis is 1x10 4 LoD was observed in copies.

[0197] Minimum detection limit (LOD) evaluation results, Ct 34, 1x10 4 The sensitivity was confirmed to be good as it was confirmed as copies. However, the results for some pathogens may vary depending on the pathogen.

[0198] <Example 9> Performance Verification - Panel Stability Test Evaluation

[0199] To verify the stability of the manufactured prototype panels, accelerated aging tests were performed under harsh temperature conditions for a short period of time equivalent to the actual storage period (Table 17).

[0200] No. Product Components Ambient Temperature (TRT) Accelerated Aging Temperature (TAA) 1 Respiratory Syndrome Panel cDNA Synthesis Kit - 20℃ 20℃ 2 Library Preparation #1 - 20℃ 20℃ 3 Library Preparation #2 - 20℃ 20℃ 4 CeleMag Clean-up Bead 4℃ 44℃ 5 CLM Polymerase - 20℃ 20℃ 6 Target Capture Solution #1 - 20℃ 20℃ 7 Target Capture Solution #2 RT 65℃ 8 Target Capture Solution #3 - 80℃ - 40℃ 9 CeleMag Streptavidin Bead 4℃ 44℃ Accelerated Aging Conditions Storage Period (Months) Accelerated Aging Time (Days) Accelerated Aging Time (AAT) = Set Expiration Date / AAF 6 1 2 1 2 3 Accelerated Aging Factor (AAF) = Q 10^(TAA - TRT / 10) Q 10 = 2, aging coefficient TAA = accelerated aging temperature (accelerated storage temperature) TRT = ambient temperature (actual storage temperature)

[0201] Sample name Pathogen form Actual storage period 6 months (accelerated aging time 12 days) Actual storage period 12 months (accelerated aging time 23 days) Detected pathogen 10x coverage (%) Detected pathogen 10x coverage (%) Mumps orthorubulavirus RNA virus Mumps orthorubulavirus 99.92 Mumps orthorubulavirus 99.91 SARS-CoV-2 RNA virus SARS-CoV-299.59 SARS-CoV-299.59 Francisella tularensis Bacteria Francisella tularensis 99.68 Francisella tularensis 99.68

[0202] As a result of the test, the product was stored under harsh conditions 40℃ higher than the actual storage temperature, and the results were consistent on the 12th day, which corresponds to the actual storage period of 6 months, and the 23rd day, which corresponds to the actual storage period of 12 months, so the product's usability period was set to 12 months.

Claims

1. In a probe set for simultaneous detection of respiratory syndrome pathogens including sequence numbers 1 to 37966, Sequence numbers 1 to 433 are for the detection of Bacillus anthracis, Sequence numbers 434 to 766 are for the detection of Blastomyces dermatitidis, Sequence numbers 767 to 1122 are for detection of Bordetella pertussis, Sequence numbers 1123 to 1490 are for the detection of Chlamydia pneumoniae, Sequence numbers 1491 to 1863 are for the detection of Chlamydia psittaci, Sequence numbers 1864 to 2266 are for the detection of Coccidioides immitis, Sequence numbers 2267 to 2665 are for the detection of Coccidioides posadasii, Sequence numbers 2666 to 3022 are for the detection of Corynebacterium diphtheria, Sequence numbers 3023 to 3409 are for the detection of Coxiella burnetii, Sequence numbers 3410 to 3778 are for the detection of Francisella tularensis. Sequence numbers 3779 to 4155 are for the detection of Haemophilus influenzae, Sequence numbers 4156 to 4544 are for the detection of Histoplasma capsulatum, Sequence numbers 4545 to 4912 are for detection of Legionella pneumophila, Sequence numbers 4913 to 5318 are for the detection of Mycobacterium avium. Sequence numbers 5319 to 5734 are for the detection of Mycobacterium intracellulare, Sequence numbers 5735 to 6230 are for the detection of Mycobacterium kansassi, Sequence numbers 6231 to 6652 are for the detection of Mycobacterium tuberculosis, Sequence numbers 6653 to 7070 are for the detection of Mycobacteroides abscessus, Sequence numbers 7071 to 7421 are for the detection of Mycoplasmoides pneumoniae, Sequence numbers 7422 to 7784 are for the detection of Neisseria meningitidis. Sequence numbers 7785 to 8168 are for the detection of Streptococcus pneumoniae, Sequence numbers 8169 to 8539 are for the detection of Streptococcus pyogenes. Sequence numbers 8540 to 8978 are for detection of Yersinia pestis, Sequence numbers 8979 to 9441 are for the detection of Coronavirus 229E. Sequence numbers 9442 to 9935 are for detection of Coronavirus HKU1, Sequence numbers 9936 to 10394 are for the detection of Coronavirus NL63. Sequence numbers 10395 to 10906 are for the detection of Coronavirus OC43, Sequence numbers 10907 to 14833 are for detection of cytomegalovirus, Sequence numbers 14834 to 17628 are for detection of Epstein-Barr virus, Sequence numbers 17629 to 17827 are for detection of Hantavirus. Sequence numbers 17828 to 18130 are for detection of Hendravirus, Sequence numbers 18131 to 18432 are for the detection of Henipavirus, Sequence numbers 18433 to 19011 are for the detection of human adenovirus type 14, Sequence numbers 19012 to 19610 are for the detection of human adenovirus type 1. Sequence numbers 19611 to 20199 are for the detection of human adenovirus type 21, Sequence numbers 20200 to 20797 are for the detection of human adenovirus type 2. Sequence numbers 20798 to 21384 are for the detection of human adenovirus type 3, Sequence numbers 21385 to 21983 are for the detection of human adenovirus type 4. Sequence numbers 21984 to 22562 are for the detection of human adenovirus type 55, Sequence numbers 22563 to 23160 are for the detection of human adenovirus type 5. Sequence numbers 23161 to 23747 are for the detection of human adenovirus type 7, Sequence numbers 23748 to 24330 are for the detection of human adenovirus type 8. Sequence numbers 24331 to 24898 are for detection of human adenovirus type, Sequence numbers 24899 to 24989 are for the detection of human bocavirus 1. Sequence numbers 24990 to 25074 are for detection of human bocavirus 2, Sequence numbers 25075 to 25161 are for the detection of human bocavirus 3. Sequence numbers 25162 to 25246 are for the detection of human bocavirus 4, Sequence numbers 25247 to 25369 are for the detection of human enterovirus CV-A21, Sequence numbers 25370 to 25492 are for the detection of human enterovirus EV-A71, Sequence numbers 25493 to 25615 are for the detection of human enterovirus EV-C104 (Human enterovirus EV-C105). Sequence numbers 25616 to 25736 are for the detection of human enterovirus EV-C105, Sequence numbers 25737 to 25858 are for the detection of human enterovirus EV-C109, Sequence numbers 25859 to 25980 are for detection of human enterovirus EV-C117, Sequence numbers 25981 to 26102 are for the detection of human enterovirus EV-C118, Sequence numbers 26103 to 26224 are for the detection of human enterovirus EV-D68, Sequence numbers 26225 to 28915 are for the detection of human herpesvirus 6, Sequence numbers 28916 to 29137 are for the detection of human metapneumovirus A, Sequence numbers 29138 to 29358 are for the detection of human metapneumovirus B, Sequence numbers 29359 to 29480 are for the detection of human parechovirus, Sequence numbers 29481 to 29695 are for detection of influenza virus A-H1N1, Sequence numbers 29696 to 29909 are for detection of influenza virus A-H3N2, Sequence numbers 29910 to 30131 are for detection of influenza virus A. Sequence numbers 30132 to 30320 are for detection of influenza virus B-victoria, Sequence numbers 30321 to 30556 are for detection of influenza virus B. Sequence numbers 30557 to 30774 are for detection of influenza virus B-yamagata, Sequence numbers 30775 to 30986 are for detection of influenza virus C, Sequence numbers 30987 to 31196 are for detection of influenza virus D, Sequence numbers 31197 to 31460 are for detection of measles morbillivirus, Sequence numbers 31461 to 31962 are for detection of MERS-CoV, Sequence numbers 31963 to 32218 are for the detection of mumps orthorubulavirus. Sequence numbers 32219 to 32522 are for detection of Nipahvirus, Sequence numbers 32523 to 32781 are for the detection of Parainfluenza virus 1, Sequence numbers 32782 to 33041 are for detection of Parainfluenza virus 2, Sequence numbers 33042 to 33298 are for the detection of Parainfluenza virus 3, Sequence numbers 33299 to 33582 are for the detection of parainfluenza virus 4a, Sequence numbers 33583 to 33868 are for the detection of parainfluenza virus 4b, Sequence numbers 33869 to 34121 are for detection of respiratory syncytial virus A, Sequence numbers 34122 to 34374 are for detection of respiratory syncytial virus B. Sequence numbers 34375 to 34493 are for detection of Rhinovirus A, Sequence numbers 34494 to 34614 are for detection of Rhinovirus B. Sequence numbers 34615 to 34732 are for detection of Rhinovirus C, Sequence numbers 34733 to 34894 are for the detection of Rubivirus rubellae, Sequence numbers 34895 to 35392 are for detection of SARS-CoV-2, Sequence numbers 35393 to 35887 are for detection of SARS-CoV, Sequence numbers 35888 to 37966 are probe sets for simultaneous detection of respiratory syndrome pathogens for the detection of Varicella zoster virus.

2. In the first paragraph, the probe set comprises SEQ ID NOs: 1 to 433; SEQ ID NOs: 434 to 766; SEQ ID NOs: 767 to 1122; SEQ ID NOs: 1123 to 1490; SEQ ID NOs: 1491 to 1863; SEQ ID NOs: 1864 to 2266; SEQ ID NOs: 2267 to 2665; SEQ ID NOs: 2666 to 3022; SEQ ID NOs: 3023 to 3409; SEQ ID NOs: 3410 to 3778; SEQ ID NOs: 3779 to 4155; SEQ ID NOs: 4156 to 4544; SEQ ID NOs: 4545 to 4912; SEQ ID NOs: 4913 to 5318; SEQ ID NOs: 5319 to 5734; SEQ ID NOs: 5735 to 6230; SEQ ID NOs: 6231 to 6652; A probe set for simultaneous detection of respiratory syndrome pathogens, comprising sequence numbers 6653 to 7070; sequence numbers 7071 to 7421; sequence numbers 7422 to 7784; sequence numbers 7785 to 8168; sequence numbers 8169 to 8539; and sequence numbers 8540 to 8978.

3. In the first paragraph, the probe set comprises SEQ ID NOs: 8979 to 9441; SEQ ID NOs: 9442 to 9935; SEQ ID NOs: 9936 to 10394; SEQ ID NOs: 10395 to 10906; SEQ ID NOs: 10907 to 14833; SEQ ID NOs: 14834 to 17628; SEQ ID NOs: 17629 to 17827; SEQ ID NOs: 17828 to 18130; SEQ ID NOs: 18131 to 18432; SEQ ID NOs: 18433 to 19011; SEQ ID NOs: 19012 to 19610; SEQ ID NOs: 19611 to 20199; SEQ ID NOs: 20200 to 20797; SEQ ID NOs: 20798 to 21384; SEQ ID NOs: 21385 to 21983; SEQ ID NOs: 21984 to 22562; SEQ ID NOs: 22563 to 23160; SEQ ID NOs: 23161 to 23747; SEQ ID NOs: 23748 to 24330; SEQ ID NOs: 24331 to 24898; SEQ ID NOs: 24899 to 24989; SEQ ID NOs: 24990 to 25074; SEQ ID NOs: 25075 to 25161; SEQ ID NOs: 25162 to 25246; SEQ ID NOs: 25247 to 25369; SEQ ID NOs: 25370 to 25492; SEQ ID NOs: 25493 to 25615; SEQ ID NOs: 25616 to 25736; SEQ ID NOs: 25737 to 25858; SEQ ID NOs: 25859 to 25980; SEQ ID NOs: 25981 to 26102; SEQ ID NOs: 26103 to 26224; SEQ ID NOs: 26225 to 28915; SEQ ID NOs: 28916 to 29137; SEQ ID NOs: 29138 to 29358; SEQ ID NOs: 29359 to 29480; SEQ ID NOs: 29481 to 29695; SEQ ID NOs: 29696 to 29909; SEQ ID NOs: 29910 to 30131; SEQ ID NOs: 30132 to 30320; SEQ ID NOs: 30321 to 30556; SEQ ID NOs: 30557 to 30774; SEQ ID NOs: 30775 to 30986; SEQ ID NOs: 30987 to 31196; SEQ ID NOs: 31197 to 31460; SEQ ID NOs: 31461 to 31962;SEQ ID NOs: 31963 to 32218; SEQ ID NOs: 32219 to 32522; SEQ ID NOs: 32523 to 32781; SEQ ID NOs: 32782 to 33041; SEQ ID NOs: 33042 to 33298; SEQ ID NOs: 33299 to 33582; SEQ ID NOs: 33583 to 33868; SEQ ID NOs: 33869 to 34121; SEQ ID NOs: 34122 to 34374; SEQ ID NOs: 34375 to 34493; SEQ ID NOs: 34494 to 34614; SEQ ID NOs: 34615 to 34732; SEQ ID NOs: 34733 to 34894; SEQ ID NOs: 34895 to 35392; SEQ ID NOs: 35393 to 35887; A probe set for simultaneous detection of respiratory syndrome pathogens comprising sequence numbers 35888 to 37966; 4. In the first paragraph, the probe set comprises SEQ ID NO: 1 to 433; SEQ ID NO: 767 to 1122; SEQ ID NO: 1491 to 1863; SEQ ID NO: 2666 to 3022; SEQ ID NO: 3023 to 3409; SEQ ID NO: 3410 to 3778; SEQ ID NO: 3779 to 4155; SEQ ID NO: 4545 to 4912; SEQ ID NO: 6231 to 6652; SEQ ID NO: 7071 to 7421; SEQ ID NO: 7422 to 7784; SEQ ID NO: 7785 to 8168; SEQ ID NO: 8169 to 8539; and SEQ ID NO: 8540 to 8978.

5. In the first paragraph, the probe set comprises SEQ ID NOs: 8979 to 9441; SEQ ID NOs: 9936 to 10394; SEQ ID NOs: 10395 to 10906; SEQ ID NOs: 10907 to 14833; SEQ ID NOs: 17629 to 17827; SEQ ID NOs: 19012 to 19610; SEQ ID NOs: 20200 to 20797; SEQ ID NOs: 20798 to 21384; SEQ ID NOs: 21385 to 21983; SEQ ID NOs: 21984 to 22562; SEQ ID NOs: 22563 to 23160; SEQ ID NOs: 23161 to 23747; SEQ ID NOs: 23748 to 24330; SEQ ID NOs: 24331 to 24898; SEQ ID NOs: 24899 to 24989; SEQ ID NOs: 25370 to 25492; SEQ ID NOs: 26103 to 26224; SEQ ID NOs: 29138 to 29358; SEQ ID NOs: 29359 to 29480; SEQ ID NOs: 29481 to 29695; SEQ ID NOs: 29696 to 29909; SEQ ID NOs: 30132 to 30320; SEQ ID NOs: 30321 to 30556; SEQ ID NOs: 30557 to 30774; SEQ ID NOs: 31197 to 31460; SEQ ID NOs: 31461 to 31962; SEQ ID NOs: 31963 to 32218; SEQ ID NOs: 32523 to 32781; SEQ ID NOs: 32782 to 33041; A probe set for simultaneous detection of respiratory syndrome pathogens, comprising SEQ ID NOs: 33042 to 33298; SEQ ID NOs: 33299 to 33582; SEQ ID NOs: 33583 to 33868; SEQ ID NOs: 33869 to 34121; SEQ ID NOs: 34122 to 34374; SEQ ID NOs: 34375 to 34493; SEQ ID NOs: 34494 to 34614; SEQ ID NOs: 34615 to 34732; SEQ ID NOs: 34733 to 34894; SEQ ID NOs: 34895 to 35392; SEQ ID NOs: 35393 to 35887; and SEQ ID NOs: 35888 to 37966.

6. A probe set for simultaneous detection of respiratory syndrome pathogens according to any one of paragraphs 1 to 5, including Bacillus anthracis, Blastomyces dermatitidis, Bordetella pertussis, Chlamydia pneumoniae, Chlamydia psittaci, Coccidioides immitis, Coccidioides posadasii, Corynebacterium diphtheria, Coxiella burnetii, Francisella tularensis, Haemophilus influenzae, Histoplasma capsulatum, and Legionella. Legionella pneumophila, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansassi, Mycobacterium tuberculosis, Mycobacterium abscessus, Mycoplasmoides pneumoniae, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes, Yersinia pestis, Coronavirus 229E, Coronavirus HKU1, Coronavirus NL63, Coronavirus OC43,Cytomegalovirus, Epstein-Barr virus, Hantavirus, Hendravirus, Henipavirus, Human adenovirus type 14, Human adenovirus type 1, Human adenovirus type 21, Human adenovirus type 2, Human adenovirus type 3, Human adenovirus type 4, Human adenovirus type 55, Human adenovirus type 5, Human adenovirus type 7, Human adenovirus type 8, Human Adenovirus (Human adenovirus type), Human bocavirus 1, Human bocavirus 2, Human bocavirus 3, Human bocavirus 4, Human enterovirus CV-A21, Human enterovirus EV-A71, Human enterovirus EV-C104, Human enterovirus EV-C105, Human enterovirus EV-C105, Human enterovirus EV-C109, Human enterovirus EV-C117,Human enterovirus EV-C118, Human enterovirus EV-D68, Human herpesvirus 6, Human metapneumovirus A, Human metapneumovirus B, Human parechovirus, Influenza virus A-H1N1, Influenza virus A-H3N2, Influenza virus A, Influenza virus B-victoria, Influenza virus B, Influenza virus B-yamagata, Influenza virus C, Influenza virus D, Measles morbillivirus, MERS-CoV, Mumps orthorubulavirus, Nipahvirus, Parainfluenza virus 1, Parainfluenza virus 2, Parainfluenza virus 3, Parainfluenza virus 4a, Parainfluenza virus 4b, Respiratory Syncytial virus A, Respiratory Syncytial virus B, Rhinovirus A,Composition for simultaneous detection of Rhinovirus B, Rhinovirus C, Rubivirus rubellae, SARS-CoV-2, SARS-CoV and Varicella zoster virus, 7. A composition for diagnosing respiratory syndrome, comprising a probe set for simultaneous detection of respiratory syndrome causative pathogens according to any one of claims 1 to 5.

8. In paragraph 7, the respiratory syndrome is anthrax, Q fever, tularemia, plague, pertussis, diphtheria, scarlet fever, legionellosis, tuberculosis, Haemophilus influenzae infection, meningococcal meningitis, pneumococcal infection, Mycoplasma pneumoniae infection, Chlamydia pneumoniae infection, psittacosis, blastomycosis, coccidioidomycosis, histoplasmosis, nontuberculous mycobacterial infection, measles, mumps, rubella, adenovirus infection, bocavirus infection, coronavirus infection, severe acute respiratory syndrome, Middle East respiratory syndrome, coronavirus disease-19, metapneumovirus infection, respiratory syncytial virus infection, influenza virus infection, parainfluenza virus infection, rhinovirus infection, chickenpox, Epstein-Barr virus infection, cytomegalovirus infection, herpesvirus 6 A composition for diagnosing respiratory syndrome, characterized by at least one selected from the group consisting of infectious diseases, hemorrhagic fever with renal syndrome, enterovirus infection, parechovirus infection, Nipah virus infection, Hennipah virus infection, and Hendra virus infection.

9. A kit for diagnosing respiratory syndrome, including a composition for diagnosing respiratory syndrome and instructions for use in accordance with Article 7.

10. In paragraph 9, the respiratory syndrome is anthrax, Q fever, tularemia, plague, pertussis, diphtheria, scarlet fever, legionellosis, tuberculosis, Haemophilus influenzae infection, meningococcal meningitis, pneumococcal infection, Mycoplasma pneumoniae infection, Chlamydia pneumoniae infection, psittacosis, blastomycosis, coccidioidomycosis, histoplasmosis, nontuberculous mycobacterial infection, measles, mumps, rubella, adenovirus infection, bocavirus infection, coronavirus infection, severe acute respiratory syndrome, Middle East respiratory syndrome, coronavirus disease-19, metapneumovirus infection, respiratory syncytial virus infection, influenza virus infection, parainfluenza virus infection, rhinovirus infection, chickenpox, Epstein-Barr virus infection, cytomegalovirus infection, A diagnostic kit for respiratory syndrome characterized by at least one selected from the group consisting of herpes virus 6 infection, hemorrhagic fever with renal syndrome, enterovirus infection, parechovirus infection, Nipah virus infection, Hennipa virus infection, and Hendra virus infection.

11. Step of preparing isolated DNA or RNA sample; A step of reacting the separated DNA or RNA sample using a probe set for simultaneous detection of respiratory syndrome pathogens of any one of claims 1 to 5; and Bacillus anthracis, Blastomyces dermatitidis, Bordetella pertussis, Chlamydia pneumoniae, Chlamydia psittaci, Coccidioides immitis, Coccidioides posadasii, Corynebacterium diphtheria, Coxiella burnetii, Francisella tularensis, Haemophilus influenzae, Histoplasma capsulatum, Legionella pneumophila, including a step of obtaining a detection result. Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansassi, Mycobacterium tuberculosis, Mycobacterium abscessus, Mycoplasmoides pneumoniae, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes, Yersinia pestis, Coronavirus 229E, Coronavirus HKU1, Coronavirus NL63, Coronavirus OC43, Cytomegalovirus, Epstein-Barr virus (Epstein-Barr virus),Hantavirus, Hendravirus, Henipavirus, Human adenovirus type 14, Human adenovirus type 1, Human adenovirus type 21, Human adenovirus type 2, Human adenovirus type 3, Human adenovirus type 4, Human adenovirus type 55, Human adenovirus type 5, Human adenovirus type 7, Human adenovirus type 8, Human adenovirus type, Human bocavirus 1, Human Human bocavirus 2, Human bocavirus 3, Human bocavirus 4, Human enterovirus CV-A21, Human enterovirus EV-A71, Human enterovirus EV-C104, Human enterovirus EV-C105, Human enterovirus EV-C105, Human enterovirus EV-C109, Human enterovirus EV-C117, Human enterovirus EV-C118,Human enterovirus EV-D68, Human herpesvirus 6, Human metapneumovirus A, Human metapneumovirus B, Human parechovirus, Influenza virus A-H1N1, Influenza virus A-H3N2, Influenza virus A, Influenza virus B-victoria, Influenza virus B, Influenza virus B-yamagata, Influenza virus C, Influenza virus D, Measles morbillivirus morbillivirus), MERS-CoV, Mumps orthorubulavirus, Nipahvirus, Parainfluenza virus 1, Parainfluenza virus 2, Parainfluenza virus 3, Parainfluenza virus 4a, Parainfluenza virus 4b, Respiratory Syncytial virus A, Respiratory Syncytial virus B, Rhinovirus A, Rhinovirus B, Rhinovirus C,Method for simultaneous detection of Rubivirus rubellae, SARS-CoV-2, SARS-CoV, and Varicella zoster virus.

12. In paragraph 11, the detection sample is characterized by being isolated from feces, blood, serum, urine, sputum or biological tissue, Bacillus anthracis, Blastomyces dermatitidis, Bordetella pertussis, Chlamydia pneumoniae, Chlamydia psittaci, Coccidioides immitis, Coccidioides posadasii, Corynebacterium diphtheria, Coxiella burnetii, Francisella tularensis, Haemophilus influenzae, Histoplasma capsulatum capsulatum), Legionella pneumophila, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansassi, Mycobacterium tuberculosis, Mycobacterium abscessus, Mycoplasmoides pneumoniae, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes, Yersinia pestis, Coronavirus 229E, Coronavirus HKU1, Coronavirus NL63, Coronavirus OC43 OC43),Cytomegalovirus, Epstein-Barr virus, Hantavirus, Hendravirus, Henipavirus, Human adenovirus type 14, Human adenovirus type 1, Human adenovirus type 21, Human adenovirus type 2, Human adenovirus type 3, Human adenovirus type 4, Human adenovirus type 55, Human adenovirus type 5, Human adenovirus type 7, Human adenovirus type 8, Human Adenovirus (Human adenovirus type), Human bocavirus 1, Human bocavirus 2, Human bocavirus 3, Human bocavirus 4, Human enterovirus CV-A21, Human enterovirus EV-A71, Human enterovirus EV-C104, Human enterovirus EV-C105, Human enterovirus EV-C105, Human enterovirus EV-C109, Human enterovirus EV-C117,Human enterovirus EV-C118, Human enterovirus EV-D68, Human herpesvirus 6, Human metapneumovirus A, Human metapneumovirus B, Human parechovirus, Influenza virus A-H1N1, Influenza virus A-H3N2, Influenza virus A, Influenza virus B-victoria, Influenza virus B, Influenza virus B-yamagata, Influenza virus C, Influenza virus D, Measles morbillivirus, MERS-CoV, Mumps orthorubulavirus, Nipahvirus, Parainfluenza virus 1, Parainfluenza virus 2, Parainfluenza virus 3, Parainfluenza virus 4a, Parainfluenza virus 4b, Respiratory Syncytial virus A, Respiratory Syncytial virus B, Rhinovirus A,Method for simultaneous detection of Rhinovirus B, Rhinovirus C, Rubivirus rubellae, SARS-CoV-2, SARS-CoV, and Varicella zoster virus.

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