tetano
Editor, Senior Moderator
Viruses
. 2023 Apr 17;15(4):983.
doi: 10.3390/v15040983. Detecting the Neuraminidase R294K Mutation in Avian Influenza A (H7N9) Virus Using Reverse Transcription Droplet Digital PCR Method
Xiuyu Lou[SUP] 1 [/SUP], Hao Yan[SUP] 1 [/SUP], Lingxuan Su[SUP] 1 [/SUP], Yi Sun[SUP] 1 [/SUP], Xinyin Wang[SUP] 1 [/SUP], Liming Gong[SUP] 1 [/SUP], Yin Chen[SUP] 1 [/SUP], Zhen Li[SUP] 1 [/SUP], Zhongbiao Fang[SUP] 2 [/SUP], Haiyan Mao[SUP] 1 [/SUP], Keda Chen[SUP] 2 [/SUP], Yanjun Zhang[SUP] 1 [/SUP]
Affiliations
The R294K mutation in neuraminidase (NA) causes resistance to oseltamivir in the avian influenza virus H7N9. Reverse transcription droplet digital polymerase chain reaction (RT-dd PCR) is a novel technique for detecting single-nucleotide polymorphisms. This study aimed to develop an RT-dd PCR method for detecting the R294K mutation in H7N9. Primers and dual probes were designed using the H7N9 NA gene and the annealing temperature was optimized at 58.0 °C. The sensitivity of our RT-dd PCR method was not significantly different from that of RT-qPCR (p = 0.625), but it could specifically detect R294 and 294K in H7N9. Among 89 clinical samples, 2 showed the R294K mutation. These two strains were evaluated using a neuraminidase inhibition test, which revealed that their sensitivity to oseltamivir was greatly reduced. The sensitivity and specificity of RT-dd PCR were similar to those of RT-qPCR and its accuracy was comparable to that of NGS. The RT-dd PCR method had the advantages of absolute quantitation, eliminating the need for a calibration standard curve, and being simpler in both experimental operation and result interpretation than NGS. Therefore, this RT-dd PCR method can be used to quantitatively detect the R294K mutation in H7N9.
Keywords: R294K mutation; RT-droplet digital PCR; absolute quantitation; avian influenza virus H7N9.
. 2023 Apr 17;15(4):983.
doi: 10.3390/v15040983. Detecting the Neuraminidase R294K Mutation in Avian Influenza A (H7N9) Virus Using Reverse Transcription Droplet Digital PCR Method
Xiuyu Lou[SUP] 1 [/SUP], Hao Yan[SUP] 1 [/SUP], Lingxuan Su[SUP] 1 [/SUP], Yi Sun[SUP] 1 [/SUP], Xinyin Wang[SUP] 1 [/SUP], Liming Gong[SUP] 1 [/SUP], Yin Chen[SUP] 1 [/SUP], Zhen Li[SUP] 1 [/SUP], Zhongbiao Fang[SUP] 2 [/SUP], Haiyan Mao[SUP] 1 [/SUP], Keda Chen[SUP] 2 [/SUP], Yanjun Zhang[SUP] 1 [/SUP]
Affiliations
- PMID: 37112963
- DOI: 10.3390/v15040983
The R294K mutation in neuraminidase (NA) causes resistance to oseltamivir in the avian influenza virus H7N9. Reverse transcription droplet digital polymerase chain reaction (RT-dd PCR) is a novel technique for detecting single-nucleotide polymorphisms. This study aimed to develop an RT-dd PCR method for detecting the R294K mutation in H7N9. Primers and dual probes were designed using the H7N9 NA gene and the annealing temperature was optimized at 58.0 °C. The sensitivity of our RT-dd PCR method was not significantly different from that of RT-qPCR (p = 0.625), but it could specifically detect R294 and 294K in H7N9. Among 89 clinical samples, 2 showed the R294K mutation. These two strains were evaluated using a neuraminidase inhibition test, which revealed that their sensitivity to oseltamivir was greatly reduced. The sensitivity and specificity of RT-dd PCR were similar to those of RT-qPCR and its accuracy was comparable to that of NGS. The RT-dd PCR method had the advantages of absolute quantitation, eliminating the need for a calibration standard curve, and being simpler in both experimental operation and result interpretation than NGS. Therefore, this RT-dd PCR method can be used to quantitatively detect the R294K mutation in H7N9.
Keywords: R294K mutation; RT-droplet digital PCR; absolute quantitation; avian influenza virus H7N9.