Virus type
Family
Basic clinical symptoms
Diagnostic methods
Influenza virus type A and B
Orthomyxoviridae
Fever, chills, muscle pain and headache, rhinitis, conjunctivitis, and inflammation of the tonsils, throat, and larynx.
Molecular methods, virus isolation in chick embryo or MDCK cell culture, antigen detection using IF immunofluorescence test, ELISA. Serological methods such as hemagglutination inhibition reaction OZHA.
Human parainfluenza virus type 1–4
Paramyxoviridae
Fever, cough, hoarseness, upper respiratory tract infection, bronchitis, and pneumonia.
Molecular methods, isolation in a continuous human cell line, such as HeLa-CPE; after 2–10 days antigen detection with F test, OZHA serological methods, and ELISA.
Human respiratory syncytial virus type A and B
Paramyxoviridae
Fever, upper respiratory tract inflammation, bronchitis, and pneumonia.
Molecular methods, isolation in a continuous human cell line, such as HeLa-CPE; after 2–10 days antigen detection with F test, OZHA serological methods, and ELISA.
Human rhinovirus type A–C
Picornaviridae
Sneezing, runny nose, cough, sore throat, headache, and less frequently fever.
Benign course and thus, frequently only clinical diagnosis or research using molecular biology techniques.
Human adenovirus
Adenoviridae
Chills, fatigue, high temperature, runny nose, dry cough, and inflammation of the glands in the neck.
Molecular methods. Isolation of the virus in the cell line HeLa or Hep-2, antigen detection with IF test.
Human coronavirus
Coronaviridae
Runny nose, sneezing, sore throat, high temperature, chills, headache, and inflammation of the lymph nodes.
Poorly replicating virus in cell culture, requiring human embryonic tracheal organ cultures or nasal epithelium; therefore molecular methods are recommended.
Human enterovirus
Picornaviridae
Pharyngitis, pneumonia.
Molecular methods, virus isolation in cell culture, serological methods, such as neutralization reaction.
Human metapneumovirus
Paramyxoviridae
Exacerbations of chronic inflammatory diseases of the respiratory system in children and adults.
Molecular methods.
Human bocavirus
Parvoviridae
Exacerbation of respiratory system diseases especially in infants and young children.
Molecular methods.
2 Methods
The material consisted of swabs from the nose and throat collected from patients during the ear, nose, and throat (ENT) emergency care in the period January–February 2013 in the Military Medical Institute in Warsaw, Poland (14 samples), and of the specimens which were accepted for tests for the detection of respiratory viruses by the Laboratory of Influenza Virus Research, National Center for Influenza in the National Institute of Public Health-National Institute of Hygiene in the season 2012/2013 (10 samples). The material was collected no later than 5 days after the onset of clinical symptoms according to the accepted criteria (WHO 2011).
2.1 Isolation of Viral RNA
The isolation of RNA is a template used in further polymerase chain reaction (PCR) studies to detect the presence of genetic material of viruses that cause respiratory tract infections. For this purpose QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) was used. The kit is designed to isolate RNA from clinical samples. A single elution with the use of elution buffer is sufficient to recover at least 90 % of the viral RNA from the QIAamp column.
2.2 Carrying Out a PCR Reaction
The RV15 One Step ACE Detection Kit (Seegene, Seoul, South Korea) is a qualitative in vitro test for the detection of 15 types of respiratory viruses in the aspirates from nasopharynx and nasopharyngeal swabs or samples from bronchopulmonary tree lavage in patients with clinical symptoms. The kit contains a set of reagents due to which it is possible to perform a Multiplex PCR. Simplification of the two-step method results in an increased repeatability of analysis and efficiency of reaction. The kit for determining 15 respiratory viruses in a sample of a clinical material is based on a process of reverse transcription (RT) and amplification of the target DNA by PCR using Dual Priming Oligonucleotide (DPO) primers, which provides freedom in a primer design and PCR optimisation and maximizes PCR specificity and sensitivity by fundamentally blocking non-specific priming. The DPO-based multiplex assay that permits the simultaneous amplification of target sequences of 15 viruses, presented after the amplification reaction, uses e.g., electrophoresis in an agarose gel preceded by nucleic acid isolation (Kim et al. 2013).
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