Development of a real-time RT-PCR assay for detection of resistance to oseltamivir in influenza A pandemic (H1N1) 2009 virus using single nucleotide polymorphism probes

Abstract

Resistance to oseltamivir in pandemic (H1N1) 2009 influenza A virus is linked to an amino acid change from histidine (H) to tyrosine (Y) at position 275 in the neuraminidase protein (NA). A real-time one step RT-PCR assay using single nucleotide polymorphism (SNP) probes was developed to detect this mutation in respiratory specimens. The limit of detection was 47.6 copies/reaction for wild-type H275 RNA and 52.9 copies/reaction for the mutant H275Y RNA. The assay did not cross-react with other respiratory pathogens. The clinical sensitivity and specificity of the assay was compared to the gold standard Sanger sequencing method using 25 sensitive, 15 resistant and 20 negative samples. The sensitivity and specificity was 88.0% and 100% respectively with the SOIV_Osel_SEN probe designed to detect the H275 allele and 100% for the SOIV_Osel_RES probe detecting the 275Y allele. The sensitivity of the assay using nine admixtures of sensitive and resistant alleles was 88.9% and 77.8% with the SOIV_Osel_SEN probe and SOIV_Osel_RES probe respectively. The presence of mixed sensitive and resistant alleles in patient samples and mixtures of in vitro RNA were detected reproducibly. This assay can be used for screening of original samples for oseltamivir resistance without the need for culture and phenotypic testing.

A multiplex real-time PCR assay for detection of oseltamivir-resistant strains of influenza virus

“…For detection of the NA gene we used a probe labeled with LC610, and for the probe detection the 610 nm channel of LightCycler was used. In previously described tests, the authors used LNA probes [10], High-Resolution Melting [11], the Cycling probe method [18], or two probes for detection of sensitive and resistant strains [13,[19][20][21][22]. There are only a few multiplex assays for detection of influenza virus and determination of oseltamivir sensitivity/resistance, and they are based on conventional PCR [12], or need three probes in the assay [23].…”

Rethinking Approaches to Improve the Utilization of Nucleic Acid Amplification Tests for Detection and Characterization of Influenza A in Diagnostic and Reference Laboratories

“…Molecular diagnosis of NAI resistance is less straightforward as substitutions in the NA gene are generally subtype-and agent-specific with E119V, R292K and N294S substitutions common in N2 subtypes and H275Y in N1 subtypes (including H1N1 and H5N1 viruses). Furthermore, as with the pandemic H1N1 (2009) strain, resistant strains may be present in patients as either single mutations (e.g., S247N or H275Y), double mutations (e.g., S247N and H275Y) or admixtures of H275Y and H275 [69][70][71].…”

“…There are several approaches to design real-time PCR assays for the detection of SNPs. Specific probes can be designed for the detection of wild type and mutant sequences; alternatively, probes with inefficient binding can be designed leading to severely attenuated probe binding such that strains with SNPs are not detected [69,77]. Regardless of the RT-PCR reaction chemistry, benefits to the PCR-based characterization of The utilization of nucleic acid amplification tests for detection & characterization of influenza A Review SNPs include lower cost as compared with other methods such as Sanger sequencing, ability to implement in a high-throughput system, ease of interpretation and the ability to use existing real-time PCR equipment that may be routinely used as detection platforms.…”

“…Thus, in some cases, a negative result using probe-based detection does not rule out the presence of viral particles carrying a SNP as a minor component of the population. In cases of emerging resistance, where mixed genotypes can occur, genetic characterization of resistance should involve a sequencing method that is able to detect a dmixtures and viral subpopulations [69].…”

“…One novel approach to the design of real-time assays utilizes primers or probes with suboptimal binding efficiency that fail to produce a PCR signal when a SNP (i.e., a mismatch) is present; the presence of IFVA is verified based on target detection using another assay [77]. This approach has been utilized in the past to identify the H275Y mutation and often requires primers and probes to be evaluated experimentally instead of an in silico-driven approach [69,77]. Probes associated with MGB proteins such as MGB-TaqMan and MGB-Eclipse ® allow the formation of thermostable duplexes with complementary singlestranded DNA targets, allowing shorter probes and higher temperatures to be used for hybridization.…”