Immuno-PCR for detection of Giardia lamblia cysts in water.

Giardia lamblia cysts at low concentrations were detected in water samples using a highly sensitive immunological-PCR (IPCR) method. Magnetic gold particles were precoated with monoclonal anti-Giardia antibodies, and Giardia lamblia cysts ranging from 1 to 100 cysts were diluted in 500 microL of water. A biotinylated detection antibody bound to the G. lamblia cysts was then linked by a streptavidin bridge to biotinylated Giardia-reporter DNA. After extensive washing, reporter DNA was released by denaturation, transferred to PCR tubes, amplified, electrophoresed, and visualized. An optimized immuno-PCR method detected as little as five G. lamblia cysts. To further evaluate the specificity and the clinical application of this IPCR assay for G. lamblia cysts, Entamoeba histolytica and Cryptosporidium parvum were also collected and detected by IPCR. The data demonstrated that this monoclonal antibody-based IPCR method is particularly useful for analysis of environmental water samples in which the densities of G. lamblia cysts is very low, and provides a platform capable of rapid screening of samples from drinking water, wells, rivers, lakes, and recreational swimming pools for trace levels of G. lamblia cysts.

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

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.