Location: Produce Safety and Microbiology ResearchTitle: Development and evaluation of a novel in situ target-capture approach for aptamer selection of human noroviruses
|LIU, DANLEI - Shanghai Jiaotong University|
|ZHANG, ZILEI - Shanghai Jiaotong University|
|JIA, FENG - Shanghai Jiaotong University|
|WU, QINGPING - Guangdong University|
|WANG, DAPENG - Shanghai Jiaotong University|
Submitted to: Talanta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/23/2018
Publication Date: 9/24/2018
Citation: Liu, D., Zhang, Z., Jia, F., Wu, Q., Tian, P., Wang, D. 2018. Development and evaluation of a novel in situ target-capture approach for aptamer selection of human noroviruses. Talanta. 193:199-205. https://doi.org/10.1016/j.talanta.2018.09.084.
Interpretive Summary: Human noroviruses (HuNoVs) are a major cause of non-bacterial acute gastroenteritis outbreaks worldwide. It accounts for more than 60,000 of hospitalization, 400,000 emergency room visits, two million outpatient visits, and 20 million total illnesses annually. Quantitative RT-PCR (RT-qPCR) has been widely used as a gold standard for the detection of HuNoVs. Although the method with merits of speed, ease, sensitive and feasibility, it has been a challenge to distinguish infectious HuNoV from inactivated HuNoV among positive-testing samples. Detection of viral capsid protein is another approach for detection of HuNoV. Antibodies, viral receptor and aptamers are commonly used in affinity binding assays for detection of viral capsid proteins. For HuNoV, the antibody-based affinity assay is not practical due to the viral antigenetic diversity. Histo-blood group antigens (HBGAs) have been recognized as receptors for HuNoVs. Previously, we demonstrated that porcine gastric mucin (PGM) contained multiple human HBGAs and could be bound by multiple strains of HuNoVs. Aptamer-based assay has been also applied as another affinity-based assay for detection of HuNoV. Aptamers are formed from selected single-stranded oligonucleotides (DNA or RNA) which could recognize and bind to their targets by molecular shape complementarities. In a standard SELEX process, the target protein-bound sequences in the library were recovered by complicated process. In this study, in situ SELEX was used to screen DNA aptamers sequences with high affinity and specificity to P particles (GII.4) in which all the bound sequences were amplified in the hybrid binding/PCR reaction wells. Traditionally, the targets for aptamers selected by SELEX are often proteins. Compared to qRT-PCR and PCR, capsid-based assays were much less sensitive. Although the sensitivity of aptamers can specifically bind to its target at level of pico- to nanomole, at least a million viral particles are needed for protein-based assays. In addition, detection of the presence of viral capsid protein does not indicate of viral infectivity. Previously, we developed a HBGA-based in situ capture qRT-PCR (ISC-qRT-PCR) assay to estimate infectivity of Tulane virus and HuNoV. ISC-qRT-PCR uses PGM to capture viral particles followed by in situ amplification of the captured viral genomes by RT-qPCR. In this study, we adapted the newly developed ISC-qRT-PCR by replacing PGM with aptamers we developed and with published aptamer (APT-M6-2). The efficacy of ISC-qRT-PCR with aptamers (APT-ISC-qRT-PCR) was compared with original ISC-qRT-PCR with PGM (PGM-ISC-qRT-PCR) in clinical samples. We demonstrated that the selected aptamer APTL-1 was comparable to PGM and slightly superior to the reported APTM6-2 aptamer for detection of HuNoVs from clinical samples.
Technical Abstract: Human noroviruses (HuNoVs) is the primary non-bacterial pathogen causing acute gastroenteritis worldwide. Molecular approaches have been mainly used for detection of HuNoVs in clinical, environmental and food samples. Aptamer-based assay has been also applied for detection of HuNoVs through affinity binding of viral capsid proteins. In a conventional systematic evolution of ligands by exponential enrichment (SELEX) process, the target protein-bound sequences in the library were recovered by complicated process such as affinity chromatography, extraction, membrane-filtration or antibody-conjugated magnetic beads. In this study, a novel approach was applied to screen aptamers for HuNoV. The new approach incorporated an in situ capture assay and next generation sequencing (NSG) for screening the aptamers. P particles of HuNoV (GII.4) were purified and coated in Nunc Immuno-Module to capture aptamers bound to the viral capsid proteins. The unbound aptamers were easily removed by washing and the sequences with high affinity were amplified and selected by repeated in situ selection process. From the total of 30,622,226 tested sequences, two aptamers, APTL-1 and APTL-6, were finally selected to incorporate with in situ capture RT-qPCR assay for detection of HuNoVs from clinical samples. The sensitivity of these two aptamers was compared with PGM that contains well-known viral receptors, and the reported aptamers APT-M6-2. Both GI and GII HuNoVs could be detected from 5 clinical samples tested. The results demonstrated that this in situ target-capture approach for screening aptamers is practicable. The selected aptamer APTL-1 was comparable to PGM and slightly superior to the reported APTM6-2 aptamer for detection of HuNoVs from clinical samples. Keywords aptamers; norovirus; HuNoVs; in situ target-capture; detection