Seeplex rv15 onestep ace detection kit

In Yokohama City between January 2013 and June 2016, 1308 clinical specimens (throat swabs, nasal swabs, nasal secretions, and nasal aspirate fluids) were collected from patients suffering upper or lower ARIs in 16 sentinel hospitals and clinics (eight pediatric clinics, four internal medicine clinics, and four hospitals) participating in the National Epidemiological Surveillance of Infectious Diseases (NESID), instituted by the Infectious Diseases Control Law in Japan, and in eight other medical institutions (one pediatric clinic and seven hospitals) (Anon, 2010 ). There were 113 hospitals and 2962 clinics in Yokohama City, and the population in the city was ∼3.7 million. Thus, the numbers of hospitals and clinics participated in this study were ∼9.7 and ∼0.4%, respectively, in the city. Before collecting the clinical specimens in which to analyze the viruses causing ARIs, the physicians at each medical institution obtained the informed consent of the patients or their guardians. The de-identified clinical specimens were sent to Yokohama City Institute of Public Health and subjected to multiplex RT–PCR with the Seeplex^® RV15 OneStep ACE Detection kit (Seegene, Seoul, South Korea), which identifies 15 major respiratory viruses. The clinical specimens that were positive for HMPV were analyzed further.

Nucleic acids were extracted from 100 μl of respiratory specimens using the PureLink Viral RNA/DNA mini kit (Invitrogen, Carlsbad, CA). RV and other fourteen respiratory viruses were detected with the Seeplex®RV15 OneStep ACE Detection kit (Seegene, Seoul, South Korea) following the manufacturer’s instructions. This diagnostic test detects the following viruses: parainfluenza viruses 1, 2, 3, and 4, adenovirus A/B/C/D/E, human coronaviruses 229E/NL63 and HuCoV-OC43, rinovirus A/B/C, influenza A, influenza B, respiratory syncyctial virus-A and -B, human bocavirus 1/2/3/4, human metapneumovirus, and enterovirus. The RNA present in the samples positive for RV was reverse transcribed with random hexamers using standard protocols. PCR was then performed with previously described primers DK001 and DK004, which target a fragment of approximately 400 bp of the hypervariable region of the RV 5′-UTR virus genome [37 (link)]. The amplified DNA fragment was purified with the High Pure PCR Product Purification kit (Roche).