A new immunoassay of serum antibodies against Peste des petits ruminants virus using quantum dots and a lateral-flow test strip.

A fast and ultrasensitive test-strip system combining quantum dots (QDs) with a lateral-flow immunoassay strip (LFIAS) was established for detection of Peste des petits ruminants virus (PPRV) antibody. The highly luminescent water-soluble carboxyl-functionalized QDs were used as the signal output and were conjugated to streptococcal protein G (SPG), which was capable of binding to immunoglobulin G (IgG) from many species through an amide bond to capture the target PPRV IgGs. The PPRV N protein, which was immobilized on the detection zone of the test strip, was expressed by transfecting recombinant Bacmid-PPRV-N with Lipofect into Sf9 insect cells. When exposed to PPRV IgG, QD-SPG bound to PPRV N protein, resulting in the formation of a complex that subsequently produced a bright fluorescent band in response to 365 nm ultraviolet excitation. Sensitivity evaluation showed that the QD-LFIAS limit of detection (LOD) for PPRV antibody was superior to competitive enzyme-linked immunosorbent assay (c-ELISA) and the immunochromatographic strip. No cross reaction was observed when the positive sera of bluetongue virus, canine distemper virus, goat pox virus, and foot-and-mouth disease virus were tested. Further evaluation using field samples indicated that the diagnostic specificity and sensitivity of the QD-LFIAS was 99.47 and 97.67 %, respectively, with excellent agreement between QD-LFIAS and c-ELISA. The simple analysis step and objective results that can be obtained within 15 min indicate that this new method shows great promise for rapid, sensitive detection of PPRV IgG for onsite, point-of-care diagnosis and post vaccination evaluation (PVE). Graphical Abstract Ultrasensitive fluorescent QD immunochromotography in combination with recombinant PPRV N protein could be used to detect PPRV antibody in serum.

[Establish and optimization of real-time fluorescent reverse transcription loop-mediated isothermal amplification for detection of avian influenza H5 hemagglutinin gene].

H5 subtype avian influenza (AIV-H5) is a major causative agent of animalloimia a rapid and sensitive molecular biological diagnosis is crucial to the control program of AIV-H5. AIV-H5 real-time fluorescent reverse transcription loop-mediated isothermal amplification (qRT-LAMP) was established by means of heat treatment of the samples. The sensitivity, specificity and repeatability of this method were assessed and the performance of Calcein,SYBR Green I,HNB,SYTO 81 in colorimetric detection was comparatively analyzed to screen the optimum dye. The results showed the sensitivity of this method was 100 times higher than that of standard real-time fluorescent RT-PCR, and the detection limit was one copy of the gene per reaction. This method had no cross-reactivity with other common avian respiratory tract infectious disease-related pathogens such as IBV and NDV. The present study suggested Calcein was the optimum dye. Small-scale tests suggested this method was reliable for survey monitoring of AIV-H5 on the spot, indicating its potential applications in field investigation.