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Development of a Sensitive Loop-Mediated Isothermal Amplification Assay That Provides Specimen-to-Result Diagnosis of Respiratory Syncytial Virus Infection in 30 Minutes
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| Zeitschriftentitel: | Journal of Clinical Microbiology |
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| Personen und Körperschaften: | , , , , , |
| In: | Journal of Clinical Microbiology, 51, 2013, 8, S. 2696-2701 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
American Society for Microbiology
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| Schlagwörter: |
| Zusammenfassung: | <jats:title>ABSTRACT</jats:title> <jats:p> Rapid isothermal amplification methods have recently been introduced, and some of these methods offer significant advantages over PCR. The objective of this study was to develop a rapid and sensitive multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of respiratory syncytial virus subgroups A and B (RSV A and B). We designed six primers each for the matrix gene of RSV A and the polymerase gene of RSV B and developed an M-LAMP assay by using a commercially available master mix and a real-time fluorometer (Genie II; Optigene, United Kingdom) that displays real-time amplification, time to positivity, and amplicon annealing temperature ( <jats:italic> T <jats:sub>m</jats:sub> </jats:italic> ). The M-LAMP was evaluated against PCR by testing 275 nasopharyngeal (NP) specimens. The final optimized M-LAMP assay had a mean amplification time of 14.2 min (compared with 90 to 120 min for PCR) and had an analytical sensitivity of 1 genome equivalent (ge) for both RSV A and B. Using PCR as a comparator, M-LAMP had a sensitivity of 100% (81/81) and specificity of 100% (194/194). We also evaluated a 3- to 10-min specimen processing method involving vortexing with glass beads and heating to 98°C in M-swab medium (Copan Italia, Brescia, Italy) and found that this rapid processing method allowed detection of 37/41 (90.2%) of positives when we used extracted nucleic acid. In summary, the M-LAMP assay had excellent sensitivity and specificity for detecting RSV A and B in NP specimens and, when coupled with a rapid specimen preparation method, could provide a specimen-to-result diagnosis time of 30 min. </jats:p> |
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| Umfang: | 2696-2701 |
| ISSN: |
0095-1137
1098-660X |
| DOI: | 10.1128/jcm.00662-13 |