Development and evaluation of NucliSens basic kit NASBA for diagnosis of parainfluenza virus infection with 'end-point' and 'real-time' detection.
New methods for the detection of human parainfluenza viruses (HPIVs) were developed. These were based on nucleic acid sequence-based amplification (NASBA) and utilised the NucliSens Basic Kit. Primers and probes were selected from the haemagglutinin neuraminidase (HN) gene of HPIV1, HPIV2 and HPIV3, and from the phosphoprotein (P) of HPIV4a and -4b. Synthetic RNA, titrated control virus stocks and respiratory specimens (n=44) were utilised to evaluate performance of the assays. Detection of NASBA products was by probe hybridisation and electrochemiluminescence (ECL) ('end-point' detection) or using molecular beacons ('real-time' detection). The assays using ECL detection proved to be both sensitive and specific. Typically, less than or equal to 100 RNA copies or one TCID(50) input was detectable with no cross-reaction between the specific HPIV assays and other respiratory viruses. Results for clinical samples were concordant with those obtained by 'conventional' procedures by classical viral diagnostic methods. 'Real-time' detection utilised probes specific for either HPIV1 or HPIV3 with similar performance characteristics to the assays with 'end-point' detection. The feasibility of multiplexing targets together was confirmed using a combined HPIV1 and HPIV3 assay with good results for ECL and molecular beacon detection on control material and clinical samples.
Hibbitts
Sam
S
Department of Medical Microbiology, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK. hibbittssj@cardiff.ac.uk
Rahman
Amanna
A
John
Rhiannon
R
Westmoreland
Diana
D
Fox
Julie D
JD
D001483:Base Sequence; D002648:Child; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D010222:Parainfluenza Virus 1, Human / Q000235:genetics / Q000302:isolation & purification; D010223:Parainfluenza Virus 2, Human / Q000235:genetics / Q000302:isolation & purification; D010224:Parainfluenza Virus 3, Human / Q000235:genetics / Q000302:isolation & purification; D029482:Parainfluenza Virus 4, Human / Q000235:genetics / Q000302:isolation & purification; D018184:Paramyxoviridae Infections / Q000175:diagnosis* / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods* / Q000706:statistics & numerical data
D017931:DNA Primers