False-Negative Results of a Validated Real-Time PCR Protocol for Diagnosis of Newcastle Disease Due to Genetic Variability of the Matrix Gene

摘要: Newcastle disease (ND) is an OIE (World Organization for Animal Health) notifiable affecting many species of birds and causing severe economic losses in the poultry sector. The caused by virulent avian paramyxoviruses serotype 1 (APMV-1). As other RNA viruses, genetic variability APMV-1 significant. Genetic analysis has revealed existence two main clades, namely classes I II, with distinct lineages sublineages (2). Class typically includes low-virulence strains recovered from live-bird markets United States or wild waterfowl worldwide (3, 4). II comprises vast majority viruses isolated poultry, including strains. official method ND diagnosis virus isolation embryonated eggs (5). Recently, molecular tests have become more common, USDA-validated real-time reverse transcription-PCR (rRT-PCR) which widely used North America Europe. This assay targets a conserved region matrix gene correlates well study it was originally (6). However, not expected to detect both avirulent all included 6). In this we report discrepancies between results samples collected three continents. We also development improved probe resulting enhanced diagnostic performance. Virus (5) rRT-PCR (6) were done confirm presence suspect submitted OIE/FAO Reference Laboratory influenza viruses. Discrepant laboratory revealed. particular, 34 domestic birds, doves, pigeons Italy, Nepal, Mauritania, Niger yielded positive but tested negative on clinical sample isolate. identification, pathotyping, genotyping isolates confirmed sequencing hypervariable part fusion (F) (1). All identified as belonging within class II. Specifically, they clustered lineage 4 (10 Italy), 5 (9 Nepal), separate novel lineage, tentatively named 7 (15 Mauritania Niger). To investigate reasons these discrepancies, fragment (M) targeted protocol sequenced analyzed. Multiple sequence alignments several nucleotide mismatches primer regions. To verify whether primers responsible false-negative rRT-PCR, loaded acrylamide gel after analysis. Distinct specific amplification products size (121 bp) visualized (Fig. ​(Fig.1),1), indicating adequate performance demonstrating that likely cause false (i.e., absence fluorescent signal assay). FIG. 1. Sodium dodecyl sulfate-acrylamide electrophoresis products. Samples original primers/probe set Lanes: 1, 1377-1/chicken/Niger/2006; 2, 1532-14/chicken/Mauritania/2006; 3, 2407-135/avian/Nepal/2006; ... To minimize variations protocol, alternative designed primers, remained unchanged. limit number degeneracy, shortened degenerated only positions. maintain stability efficiency, locked six positions introduction modified nucleotides (locked nucleic acid [LNA] probe). Primer are illustrated Fig. ​Fig.2.2. LNA hexachloro-6-carboxyfluorescein (HEX)-GGGAcRgcHtGcTATcC-black hole quencher (BHQ) 3′ (lowercase letters indicate positions). FIG. 2. Matrix alignment representative illustrating sequences according (underlined). indicated ... The specificity 9 (lineages 4, 5) unrelated microorganisms such APMV serotypes 2 (7 strains), infectious bronchitis (5 (17 strains; subtypes H1 H16), pneumovirus types A B. test capable detecting strains, no cross-reactions found. analytical sensitivity new (62 × 102 copies/μl) comparable procedure On specimens, threshold cycle (CT) values similar some cases lower than CT obtained protocol. variant showing excellent broadening applicability known isolates. The diversity among may produce when relied on. Our suggest should be abandoned support importance monitoring circulating rapidly identify possible antigenic variants.