Application of heteroduplex mobility assay in quick identification of adenovirus subtypes in epidemic keratoconjunctivitis / 中华实验眼科杂志

ABSTRACT

Background The commonly used method of typing the adenovirus (AdV) for epidemic keratoconjunctivitis is direct DNA sequence.However,heteroduplex mobility assay (HMA) is found to be a faster method to identify the subtypes of adenovirus and is more conforming to the principle of cost and economic benefit.There are few studies to illustrate the application of HMA in epidemic keratoconjunctivitis.Objective This study analyzed the conserved region of hexon coding sequence and compared the outcomes between direct DNA sequence and HMA for classification of adenovirus in epidemic keratoconjunctivitis patients.The validity of HMA is evaluated by comparing the result of both studies Methods Two hundreds and fourteen patients with suspicious epidemic keratoconjunctivitis were included in Shanghai Eye Disease Prevention and Treatment Center or the clinical sites supervised by the Shanghai Prevention and Monitoring Office of Acute Hemerragic Conjunctivitis from January 2010 to December 2012.Sacconjunctival swab samples were collected from each patient under the informed consent.DNA of pathogens was extracted from the samples using QIA-amp minikit,and the conserved sequence with 366 bp at hexon protein coding region was amplified by PCR and sequenced subsequently to determine the infected adenovirus and their subtypes.These samples were simultaneously assayed by HMA,and the outcomes between DNA sequence and HMA were compared.Results Extracted DNA presented a yellow fluorescence band with the fragment size 35 kb and absorbance ratio at the wavelength 260 nm and 280 nm (A260/A280) was I.7.In the 214 samples,AdV type 1 (AdV1) was found in 4 samples,AdV2 in 33 samples,AdV3 in 15 samples,AdV4 in 12 samples,AdV8 in 19 samples,AdV19 in 15 samples and AdV37 in 8 samples.HMA showed the same outcome for the identification of AdV1,AdV2,AdV3,AdV8,AdV19 and AdV37 with direct DNA sequence.AdV4 was not feasible to HMA owing to 59.6％ (over 10％) mutation sites.Conclusions Direct DNA sequencing for conserved regions in coding sequence of hexon is an important way to identify causing-disease adenovirus subtypes in the patients with epidemic keratoconjunctivitis.HMA can offer a consistent result with the DNA sequencing,and it might be used as a suitable tool for large-scale molecular epidemiology researches.