Development of a quadruple qRT-PCR assay for simultaneous identification of highly and low pathogenic H7N9 avian influenza viruses and characterization against oseltamivir resistance

Abstract

The HA genes of the HP-H7N9 virus possessed either a KRTA, KRIA, or KRAA insertion with a nucleic acid 12 bp in size, 5′-AAA CGG A(G)C(T)T GCG-3′. [...]we designed the forward (H7-F), reverse (H7-R) primers covering this region, and a probe (H7-P-M) which can specifically recognize the specific insertion. The multiplex qRT-PCR method using DNA standards as template showed that Ct values were similar among the four targets, and the linear range was between 1 × 106 to 1 × 101 molecules, respectively. [...]the regression coefficients (R2) values were above 0.99, indicating that the assay was both accurate and precise over this range (Fig. 2). The emergence of an NAI-resistance mutation is temporally associated with a rebound of virus load, treatment failure and a poor clinical outcome [16]. [...]identification of the NAI-mutation in time may increase the cure rate for H7N9 infections. [...]appropriate laboratory methods for the continuous efforts of laboratory surveillance are of great importance [12].