All NPAs were tested using the multiplex real-time PCR Anyplex II RV16 detection kit (Seegene). This system is able to detect and differentiate DNA/RNA from 15 of the most frequent human respiratory viruses (metadata file: https://doi.org/10.6084/m9.figshare.13280435.v3 ).
Anyplex 2 rv16 detection kit
Manufactured by Seegene
Sourced in Cameroon
The Anyplex II RV16 Detection Kit is a real-time PCR-based diagnostic assay designed to detect and identify 16 common respiratory viruses, including influenza A/B, respiratory syncytial virus A/B, and human rhinovirus, among others. The kit provides a rapid and sensitive method for the simultaneous detection of these respiratory pathogens.
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29 protocols using anyplex 2 rv16 detection kit
1
Multiplex Real-time PCR for Respiratory Viruses
2
Diagnosis of Influenza and Febrile Seizures
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Influenza was diagnosed when at least one of two virological tests identified influenza A or B viruses. A commercial rapid influenza diagnostic test (RIDT) kit (Alere BinaxNOW® Influenza A & B Card, Abbott, IL, USA) and multiplex polymerase chain reaction (mPCR) test kit (Anyplex® II RV16 Detection Kit, Seegene Inc., Seoul, Korea) were used to identify RVs in a nasopharyngeal swab sample. In our hospital, mPCR tests for RVs were performed for hospitalized patients presenting with respiratory symptoms or fever without localizing signs, unless the guardian refused testing. During the influenza season, RIDTs were performed prior to mPCR testing, and mPCR tests were performed based on the attending physician’s decision. RV infection, other than influenza, was diagnosed when the mPCR test result was positive for at least one of the tested RVs (adenovirus; parainfluenza virus types 1, 2, 3, and 4; rhinovirus; bocavirus; coronavirus 229E, OC43, and NL63; enterovirus; human metapneumovirus; and respiratory syncytial virus [RSV] groups A and B) excluding the influenza virus. FS was defined as a seizure accompanied by fever (body temperature ≥38 °C) occurring in children aged between 6 and 60 months without other causes of seizures. Seizures that occurred in patients aged >60 months were defined as late-onset seizures.
3
Viral Respiratory Pathogen Surveillance
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For all febrile patients, a nasopharyngeal sample was collected, placed in 2 mL of Universal Transport Medium (COPAN Diagnostics) and stored at 4 °C before being sent weekly to the National Influenza Center (NIC) of Senegal, Institute Pasteur of Dakar (IPD). For respiratory viruses, a two-step Real-Time Reverse Transcription–Polymerase Chain Reaction (RT-PCR) was performed using the CFX96 Real-time PCR system (Bio-Rad) and Anyplex II RV16 Detection kit (Seegene), as previously described [24 (link)].
4
Comprehensive Respiratory Pathogen Assessment
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The cut-off values for interpreting an MP infection status based on the MP-specific IgM titers were assessed in accordance with the manufacturer’s instructions, as positive (IgM, > 1.1) and negative (IgM, < 0.9). Macrolide resistance was evaluated by identifying the point mutations at both positions 2063 and 2064 in domain V of the 23 S rRNA of MP by the GENECUBE system and GENECUBE Mycoplasma detection kit (Sin Corporation, Tokyo, Japan). The presence of 16 respiratory viral pathogens, including adenovirus, respiratory syncytial viruses A and B, rhinovirus, influenza viruses A and B, parainfluenza viruses 1–4, bocavirus, metapneumovirus, enterovirus, and corona viruses OC43, 229E, and NL63, was detected in nasopharyngeal swab samples by means of a PCR assay using the Anyplex II RV16 Detection kit (Seegene, Seoul, South Korea). In some of the patients with available sputum samples, the Pneumobacter PCR (Allplex Pneumobacter Assay, Seegene, Seoul, South Korea) assay was performed, which can detect Streptococcus pneumoniae, Haemophilus influenza, MP, Chlamydophila pneumoniae, Bordetella pertussis, and Legionella pneumophila.
5
Respiratory Virus Detection Protocol
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The detection of HPIV-3 and other respiratory viruses was routinely performed either by direct immunofluorescence antigen detection (D3Ultra 8™ DFA Respiratory Virus Screening & Identification Kit Diagnostic HYBRIDS, USA) or by a multiplex real-time PCR assay (Anyplex II RV16 Detection Kit, Seegene, Korea). Total nucleic acids (NA) were purified by using NucliSENS® EasyMag® (BioMérieux, Marcy l'Etoile, France) from 400 μl of respiratory specimens and eluted to 100 μl according to the manufacturer's instructions. Eluted NAs were kept at −80 °C until use.
6
Multiplex Respiratory Virus Detection
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RV16 testing was performed to detect the following viruses: adenovirus, influenza viruses A and B, RSV A, RSV B, parainfluenza viruses 1 to 4, HRV, HMPV, human enterovirus, coronavirus 229E, coronavirus NL63, coronavirus OC43, and human bocavirus. During the RV16 test, an internal control was added to each specimen to check the entire process from nucleic acid extraction to PCR, according to the manufacturer's instructions. An Anyplex II RV16 Detection Kit (Seegene) was used to detect fourteen types of RNA viruses and two types of DNA viruses, according to the manufacturer's instructions. Briefly, the assay was conducted in a final volume of 20 µL containing 8 µL of cDNA, 4 µL of 5 × RV primer, 4 µL of 8-methoxypsoralen solution, and 4 µL of 5 × master mix with the CFX96 real-time PCR detection system (Bio-Rad Laboratories Inc., Hercules, CA, USA).
7
Multiplex RT-PCR for Respiratory Viruses
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Total viral nucleic acid (DNA and RNA) was extracted from 140 μL of each clinical specimen using the PureLink Viral RNA/DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. DNA/RNA was eluted with 60 μL nuclease-free water and stored at −80°C until use. A 2-step multiplex real-time reverse transcription-polymerase chain reaction was performed with a Bio-Rad CFX-96 thermo cycler (Bio-Rad Laboratories, Singapore) and the Anyplex II RV16 Detection Kit (Seegene, Seoul, Korea) for a simultaneous testing of influenza viruses (fluA and fluB), human respiratory syncytial virus (RSVA and RSVB), human adenoviruses (HAdV), HMPV, human coronavirus (229E, NL63, OC43), human parainfluenza virus (PIV1, 2, 3, and 4), human rhinovirus (HRV), human enterovirus (HEV), and human bocavirus (HBoV).
8
Multiplex rtPCR for Respiratory Viruses
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The multiplex rtPCR was performed according to the manufacturer’s instruction (Seegene, Korea). The Anyplex™ II RV16 detection kit (Seegene, Korea) detects the following viruses: adenovirus, influenza A virus, influenza B virus, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, rhinovirus A/B/C, RSV A, RSV B, bocavirus 1/2/3/4, human metapneumovirus, coronavirus 229E, coronavirus NL63, coronavirus OC43, and enterovirus. Specimens that arrived before mid-morning from Monday to Friday were processed daily and results were provided by mid-afternoon. Specimens that arrived afterwards were processed on the following workday and reported by mid-afternoon. Dates of specimen collection and testing were available, but not date and time of reporting of results. For CRP, white blood cell count (WBC) and platelets, the most pathologic values within 3 days before and after rtPCR results were documented.
9
Multiplex RT-PCR Respiratory Virus Detection
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RSV, influenza virus and other respiratory viruses (adenovirus; bocavirus 1-4; coronavirus 229E, OC43 and NL63; human metapneumovirus; parainfluenza virus 1-4; and picornavirus) were detected by multiplex RT-PCR in nasopharyngeal swabs using Allplex Respiratory Panels 1, 2 and 3 or an Anyplex II RV 16 detection kit (Seegene, Seoul, South Korea). Any available bronchoalveolar lavage fluid samples or tracheal aspirates were also tested. Samples were tested in the virology laboratory of each participating hospital.
10
Multiplex Real-time PCR for Respiratory Viruses
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Real-time multiplex PCR with an Anyplex II RV16 Detection kit (Seegene, Seoul, Korea) using nasopharyngeal swab samples was performed, which targets 16 respiratory viruses, including adenovirus, respiratory syncytial viruses A and B, rhinovirus, influenza viruses A and B, parainfluenza viruses 1–4, bocavirus, metapneumovirus, enterovirus, and corona viruses OC43, 229E, and NL63 [2 (link)].
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