Development of a triple NanoPCR method for feline calicivirus, feline panleukopenia syndrome virus, and feline herpesvirus type I virus.

BACKGROUND: Feline calicivirus (FCV), Feline panleukopenia virus (FPV), and Feline herpesvirus type I (FHV-1) are the three most common pathogens in cats, and also are the main pathogens leading to the death of kittens. Here, by a combination of gold nanoparticles and conventional PCR, we established a novel triple NanoPCR molecular detection method for clinical detection. RESULTS: The triple NanoPCR molecular detection is able to detect 2.97 × 101copies/µL FCV recombinant copies plasmid per reaction, 2.64 × 104copies/µL FPV recombinant copies plasmid per reaction, and 2.85copies/µL FHV-1 recombinant copies plasmid per reaction at the same time. The sensitivity of each plasmid is 100 times, 10 times, and 100 times higher than conventional PCR, respectively. The clinical results showed that among the 38 samples, the positive rates of FCV, FPV, and FHV-1 in a NanoPCR test were 63.16, 31.58, and 60.53%, while in a conventional PCR were 39.47, 18.42, and 34.21%. CONCLUSIONS: In this report, it is the first time that NanoPCR assays are applied in the detection of FCV, FPV, and FHV-1 as well. This sensitive and specific NanoPCR assay can be widely used in clinical diagnosis and field monitoring of FCV, FPV, and FHV-1 infections.

A lateral flow dipstick combined with reverse transcription recombinase polymerase amplification for rapid and visual detection of the BVDV and BPIV3.

Bovine respiratory disease complex (BRDC) is a serious disease affecting feedlot cattle in China and likely other places worldwide. Bovine viral diarrhea virus (BVDV) and bovine parainfluenza virus type 3 (BPIV3) are principally responsible for causing BRDC, and are a major strain to the industrial economy. Eradication of these viruses/disease requires swift viral identification and treatment. Hence, this study established a fast and easy procedure of BVDV and BPIV3 identification that employs reverse transcription recombinase polymerase amplification (RT-RPA) and lateral flow dipstick (LFD), and uses primers and lateral flow (LF) probe targeting the 5'-UTR gene of BVDV and phosphoprotein P gene of BPIV3, respectively. Our assay was able to successfully amplify BVDV and BPIV3 RNA within 25 min at 35 °C using RT-RPA, with products visible on the LFD within 5 min at room temperature (RT). The lowest detection limits were 50 RNA molecules for BVDV and 34 RNA molecules for BPIV3 per reaction. We also demonstrated that the established dual RT-RPA LFD assay was precise and targeted, harboring excellent potential to become an onsite molecular diagnostic tool in the detection of BVDV and BPIV3. This method can detect BVDV (Pestivirus A, B) and BPIV3, and exhibit no cross-reaction with other viruses like the classical swine fever virus (CSFV) and infectious bovine rhinotracheitis virus (IBRV). The assay performance was further assessed with clinical samples, and demonstrated good performance in comparison to real-time RT-PCR (RT-qPCR). Moreover, the RT-RPA LFD assay was comparitively rapid and required minimal training.