Development of a real-time multiplex RSV detection assay for difficult respiratory samples, using ultrasone waves and MNAzyme technology.
BACKGROUND
Elderly infected with Human Respiratory Syncytial Virus (RSV) often bear low viral loads that stay below the detection limits of commercial assays. A more sensitive detection of RSV infections can improve patient management, guide containment strategies, and possibly prevent morbidity and mortality among populations most severely affected by RSV.


OBJECTIVE
To test the sensitivity for RSV detection by using an alternative extraction method in combination with a new amplification procedure.


METHODS
Nasopharyngeal washes and sputum samples (n=78) form clinical cases, and broncheo-alveolar lavages (n=27) from an experimental RSV rat model were obtained. An ultrasone-based RNA extraction method was combined with a multi-component Nucleic Acid enzymes (MNAzyme) amplification procedure for simultaneous detection of RSV-A, RSV-B, and an Internal Extraction control IEC.


RESULTS
Compared to standard real-time PCR technology, this method resulted in an increased detection sensitivity, ranging from 0.9 to 4.93 log (average 2.05+/-1.01) for RSV-A and 0.76 to 4.28 log (average 1.30+/-0.92) for RSV-B.


CONCLUSIONS
An ultrasone-based extraction method with MNAzyme amplification resulted in improved detection of RSV in different respiratory samples, including sputum. This generic method for nucleic acid extraction should be readily applicable for any other respiratory pathogen.
Nauwelaers
David
D
Virco BVBA, Generaal De Wittelaan L 11B 4, B-2800 Mechelen, Belgium. dnauwela@its.jnj.com
Vijgen
Leen
L
Atkinson
Claire
C
Todd
Alison
A
Geretti
Anna Maria
AM
Van Ranst
Marc
M
Stuyver
Lieven
L
D000818:Animals; D001483:Base Sequence; D001992:Bronchoalveolar Lavage Fluid / Q000821:virology*; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D009305:Nasopharynx / Q000821:virology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification*; D051381:Rats; D015203:Reproducibility of Results; D018113:Respiratory Syncytial Virus, Human / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013183:Sputum / Q000821:virology*
D017931:DNA Primers; D012367:RNA, Viral