A highly sensitive immuno-PCR assay for detection of H5N1 avian influenza virus

Abstract

With an aim at detecting the ultra-low concentration of avian influenza virus (AIV), a highly sensitive hybrid assay based on immunology and polymerase chain reaction was developed. The TopYield microtiter plates were coated with ten-fold serial dilutions of H5N1 subtype AIV ranging from 10 EID(50 )ml(-1)~10(-4) EID(50) ml(-1),which was recognized by mouse anti-AIV H5 monoclonal antibody (MAb) that was directly linked with reporter DNA using a heterobifunctional cross-linker. After extensive washing, the reporter DNA including a BamH I-restriction site was released by a specific enzymatic restriction, then transferred to PCR tubes, amplified, and used as the signal for detection of AIV. Under the optimized condition, MAb-based immuno-PCR (IPCR) method could measure 100 µl of AIV H5N1 with 10(-4 )EID(50) ml(-1).To evaluate the sensitivity of IPCR, the same concentration and volume of AIV H5N1 were detected by conventional RT-PCR and sandwich ELISA. The results showed that IPCR had an approximately 1,000-fold improvement over the conventional ELISA, and a 100-fold enhancement compared with RT-PCR in detection sensitivity. To further evaluate the specificity of IPCR for AIV H5 subtype, the tracheal swab specimens, taken from chickens which were infected with H9N2, and the allantoic fluid from eggs inoculated by AIV H3N2, H7N1, H9N2, were detected by IPCR. To mimic clinical samples, pharyngeal-tracheal swab specimens were collected from healthy chickens and spiked with H5N1, H5N2, H5N3 for analysis by immuno-PCR. The results demonstrated that IPCR was a highly sensitive and specific assay for AIV H5, and could be applied to clinical detection for low amount of AIV H5 subtype. This MAb-based immuno-PCR method provided a platform capable of mass screening of clinical samples for AIV H5 subtype and could serve as a model for other immuno-PCR assays.

Multiplexed detection of influenza A virus subtype H5 and H9 via quantum dot-based immunoassay

“…The assay consists of only one step: adding the sample and the QDs-Ab mixtures onto the QDs-LFIAS strip and checking the result presented by the equipment in 15 min, which eliminates the need for professional laboratory technicians and expensive equipment and permits high-throughput screening. Alternatively, nucleic acid-based detection methods were proved to be highly efficient, specific and accurate detection methods for viruses (Deng et al 2011;Grabowska et al 2014;Lai et al 2012;Payungporn et al 2006;Zhao et al 2010). …”

“…These methods are categorized based on the type of detection target: nucleic acid-based detection, virus isolation and identification, antigen detection, antibody detection, etc. (Alberini et al 2009;Chen et al 2008;Ho et al 2009;Moore et al 2010;Payungporn et al 2006;Xie et al 2006;Yang et al 2008) The most commonly used detection methods are nucleic acid-based detection, such as reverse-transcription PCR, Real-time PCR, RT-LAMP, antibody detection and antigen detection (Antarasena et al 2007;Deng et al 2011;He et al 2007;Kang et al 2010;Khurana et al 2011;Shahsavandi et al 2011;Yea et al 2010;Zhao et al 2010). However, most of these methods require specialized equipment and highly trained operators, and these detection processes are time consuming, thereby making them impractical for point-of-care (POCT) detection (Yager et al 2008 Quantum dots (QDs) have been broadly used for biological labeling and imaging due to their unique optical properties, i.e., a broad excitation spectrum and a narrow and symmetric emission peak (Lu et al 2011;Medintz et al 2005;Zhou et al 2011b).…”

Diagnosis of pulmonary and extrapulmonary tuberculosis based on detection of mycobacterial antigen 85B by immuno-PCR

“…However, it would be worthwhile to compare I-PCR versus Xpert assay on the same specimens to ensure the efficacy of our I-PCR test. Similar to this study, there are reports for the ultra-low detection of other bacterial and viral antigens using SMCC based I-PCR, for example, Clostridium botulinum neurotoxin A and H5N1 Avian influenza virus (Deng et al, 2011;Wu et al, 2001). Instead of using SMCC, another chemical linker N-succinimidyl-S-actyl-thioacetate (SATA) has been employed by Fischer et al (2007) for the detection of Staphylococcus aureus enterotoxin A and B by real-time I-PCR.…”

Novel methodologies in analysis of small molecule biomarkers and living cells

“…According to the classification of immunoassays, quantitative immuno-PCR includes direct quantitative immuno-PCR, competitive quantitative immuno-PCR, and sandwich quantitative immuno-PCR ( Fig. 1) [13,14,7].…”