Advances in Diagnostic Approaches for Viral Etiologies of Diarrhea: From the Lab to the Field

FIGURE 1. A schematic representation of diagnostic methods for enteric viruses. The diagnostic assays are classified in five major categorizes (i) cell culture, (ii) electron microscopy, (iii) serological methods, (iv) nucleic acid methods, which further have three sub-classifications, and (v) next-generation methods. The techniques are individually dealt under different sections.

FIGURE 2. A schematic representation of Immuno PCR and PCR-ELISA. There are different immuno PCR platforms available as shown in figure (A–E). The Immuno PCR format starts with an immune assay followed by PCR. Immuno PCR is similar to ELISA except terminal DNA is amplified by a PCR. (A) Sandwich format of Immuno PCR, (B) Direct format of antigen detection, (C) Phage mediated where single chain variable fragments (scFv) and DNA are carried by the phage and upon heating DNA is released from the phage, (D) Magneto Immuno PCR, where the antibody is captured on a magnetic bead with the rest of the protocol being similar to the sandwich format, (E) Nanoparticle amplified immune PCR where magnetic beads capture several antibodies which uses gold nanoparticle in a sandwich pattern to amplify DNA by PCR. PCR-ELISA starts with PCR followed by an immune assay and detecting the color production. The target gene is amplified in the presence of DIG-dUTP. Employing the avidin biotin affinity, the amplified gene is immobilized on plate and finally using substrate, enzyme and conjugate, color production is visualized.

FIGURE 3. Comparison between thermal cycling based PCR and isothermal LAMP assays employed for the detection of viruses and a glimpse on recent advances in LAMP assays. (A) LAMP does not require sophisticated instruments; a water bath is sufficient and time for completion is approximately 1 h. Results can directly be visualized by addition of SYBR Green or hydroxynapthol blue (HNB) or calcein dyes. Gel electrophoresis can also be employed where a ladder-like pattern can be visualized. Additionally, closed tube LAMP assays are being developed. (B) RT-PCR starts with RNA extraction from virus and cDNA preparation followed by repeated cycling using a sophisticated thermal cycler and procedure takes approximately 3 h to complete. Subsequently, to analyze the results, gel electrophoresis has to be carried out, which takes additional 1 h. (C) PCR starts with DNA extraction from virus followed by all cycles stated in B step and takes 3–4 h to complete the procedure. (D) Advances in LAMP, like Immunocapture LAMP (1) assays are being developed where antibodies against the virus are coated in the tubes and the LAMP mixture is in lyophilized form at the bottom of the tubes. On virus sample addition…

FIGURE 4. Next generation techniques used in the detection of enteric viruses. Recent techniques like (A) Biosensors, (B) Electrochemical sensor, (C) Microarray, (D) Aptamers/Aptasensors etc. have been employed for the detection of enteric viruses. Further, in aptamer based diagnostics: (A) Aptamer-linked immunosorbent assay, ALISA (Alike ELISA except use of antibodies), (B) SPR-aptasensors, and (C) cantilever aptasensors are in use for detecting various viruses including norovirus strains.