tetano
Editor, Senior Moderator
BMC Infect Dis. 2018 Aug 15;18(1):406. doi: 10.1186/s12879-018-3302-7.
[h=1]Development of a quadruple qRT-PCR assay for simultaneous identification of highly and low pathogenic H7N9 avian influenza viruses and characterization against oseltamivir resistance.[/h] Yang Y[SUP]1,[/SUP][SUP]2[/SUP], Li S[SUP]1,[/SUP][SUP]2[/SUP], Wong G[SUP]1,[/SUP][SUP]2[/SUP], Ma S[SUP]2[/SUP], Xu Z[SUP]1[/SUP], Zhao X[SUP]3[/SUP], Li H[SUP]3[/SUP], Xu W[SUP]3[/SUP], Zheng H[SUP]1[/SUP], Lin J[SUP]1[/SUP], Zhao Q[SUP]1[/SUP], Liu W[SUP]2[/SUP], Liu Y[SUP]1,[/SUP][SUP]4[/SUP], Gao GF[SUP]1,[/SUP][SUP]2,[/SUP][SUP]5,[/SUP][SUP]4[/SUP], Bi Y[SUP]6,[/SUP][SUP]7[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] [h=4]BACKGROUND:[/h] During the fifth wave of human H7N9 infections, a novel highly pathogenic (HP) H7N9 variant emerged with an insertion of multiple basic amino acids in the HA cleavage site. Moreover, a neuraminidase inhibitor (NAI) resistance (R292K in NA) mutation was found in H7N9 isolates from humans, poultry and the environment. In this study, we set out to develop and validate a multiplex quantitative reverse transcript polymerase chain reaction (qRT-PCR) to simultaneously detect the presence of H7N9 and further identify the HP and NAI-resistance mutations.
[h=4]METHODS:[/h] A quadruple qRT-PCR to simultaneously detect the presence of H7N9 and further identify the HP and NAI-resistance mutations was designed based on the analyses of the HA and NA genes of H7N9. This assay was further tested for specificity and sensitivity, and validated using clinical samples.
[h=4]RESULTS:[/h] The assay was highly specific and able to detect low pathogenic (LP)- or HP-H7N9 with/without the NAI-resistance mutation. The detection limit of the assay was determined to be 50 genome-equivalent copies and 2.8 ? 10[SUP]- 3[/SUP] 50% tissue culture infectious doses (TCID[SUB]50[/SUB]) of live H7N9 per reaction. Clinical validation was confirmed by commercial kits and Sanger sequencing with ten clinical samples.
[h=4]CONCLUSIONS:[/h] We developed and validated a rapid, single-reaction, one-step, quadruple real-time qRT-PCR to simultaneously detect the presence of H7N9 and further identify the HP- and NAI-resistance strains with excellent performance in specificity and sensitivity. This assay could be used to monitor the evolution of H7N9 viruses in the laboratory, field and the clinic for early-warning and the prevention of H7N9 infections.
[h=4]KEYWORDS:[/h] H7N9; Highly/low pathogenic avian influenza virus; Molecular diagnostics; NAI-resistance; Oseltamivir; Quadruple qRT-PCR
PMID: 30111290 DOI: 10.1186/s12879-018-3302-7
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[h=1]Development of a quadruple qRT-PCR assay for simultaneous identification of highly and low pathogenic H7N9 avian influenza viruses and characterization against oseltamivir resistance.[/h] Yang Y[SUP]1,[/SUP][SUP]2[/SUP], Li S[SUP]1,[/SUP][SUP]2[/SUP], Wong G[SUP]1,[/SUP][SUP]2[/SUP], Ma S[SUP]2[/SUP], Xu Z[SUP]1[/SUP], Zhao X[SUP]3[/SUP], Li H[SUP]3[/SUP], Xu W[SUP]3[/SUP], Zheng H[SUP]1[/SUP], Lin J[SUP]1[/SUP], Zhao Q[SUP]1[/SUP], Liu W[SUP]2[/SUP], Liu Y[SUP]1,[/SUP][SUP]4[/SUP], Gao GF[SUP]1,[/SUP][SUP]2,[/SUP][SUP]5,[/SUP][SUP]4[/SUP], Bi Y[SUP]6,[/SUP][SUP]7[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] [h=4]BACKGROUND:[/h] During the fifth wave of human H7N9 infections, a novel highly pathogenic (HP) H7N9 variant emerged with an insertion of multiple basic amino acids in the HA cleavage site. Moreover, a neuraminidase inhibitor (NAI) resistance (R292K in NA) mutation was found in H7N9 isolates from humans, poultry and the environment. In this study, we set out to develop and validate a multiplex quantitative reverse transcript polymerase chain reaction (qRT-PCR) to simultaneously detect the presence of H7N9 and further identify the HP and NAI-resistance mutations.
[h=4]METHODS:[/h] A quadruple qRT-PCR to simultaneously detect the presence of H7N9 and further identify the HP and NAI-resistance mutations was designed based on the analyses of the HA and NA genes of H7N9. This assay was further tested for specificity and sensitivity, and validated using clinical samples.
[h=4]RESULTS:[/h] The assay was highly specific and able to detect low pathogenic (LP)- or HP-H7N9 with/without the NAI-resistance mutation. The detection limit of the assay was determined to be 50 genome-equivalent copies and 2.8 ? 10[SUP]- 3[/SUP] 50% tissue culture infectious doses (TCID[SUB]50[/SUB]) of live H7N9 per reaction. Clinical validation was confirmed by commercial kits and Sanger sequencing with ten clinical samples.
[h=4]CONCLUSIONS:[/h] We developed and validated a rapid, single-reaction, one-step, quadruple real-time qRT-PCR to simultaneously detect the presence of H7N9 and further identify the HP- and NAI-resistance strains with excellent performance in specificity and sensitivity. This assay could be used to monitor the evolution of H7N9 viruses in the laboratory, field and the clinic for early-warning and the prevention of H7N9 infections.
[h=4]KEYWORDS:[/h] H7N9; Highly/low pathogenic avian influenza virus; Molecular diagnostics; NAI-resistance; Oseltamivir; Quadruple qRT-PCR
PMID: 30111290 DOI: 10.1186/s12879-018-3302-7
Free full text