Genetic evolutionary analysis of a strain of Senecavirus A in Anhui and the establishment of its detection method.

BACKGROUND: Senecavirus A (SVA) is the only member of the genus Senecavirus in the family Picornaviridae, and is one of the pathogens of porcine blistering disease. SVA has been reported in the United States, Canada, China, Thailand, and Colombia. METHODS: In this study, positive SVA infection was detected by RT-PCR in sick materials collected from pig farms of different sizes in Anhui Province. RESULTS: In this study, a virulent strain of SVA was successfully obtained by viral isolation on BHK21 cells and named SVA-CH-AHAU-1. Meanwhile, a simple, rapid and accurate nano-PCR method for the detection of SVA infection was established in this study, using the recombinant plasmid pClone-SVA-3D as a template. CONCLUSIONS: The complete genome of SVA-CH-AHAU-1 is 7286 bp, including a 5' non-coding region (UTR), an open reading frame (ORF) of 6546 nucleotides, encoding 2182 amino acids (aa), and a 3' UTR with Poly(A) features, and phylogenetic analysis showed that this isolate had the highest nucleotide homology (97.9 %) with the US isolate US-15-41901SD. In this study, the virulent strain SVA-CH-AHAU-1 was found to recombine in the ORF region with isolates SVA-CH-SDGT-2017 and SVA/Canada/ON/FMA-2015-0024 T2/2015. The complete genome has been submitted to GeneBank with the accession number OM654411. In addition, our results suggest that the established nano-PCR assay can be used as an economical, reliable and sensitive method for the field diagnosis of SVA method, especially in resource-limited areas.

Nano-PCR for the early detection of tomato leaf curl virus.

Nano-PCR is a potential tool for the early detection of plant viruses. In the current study, different concentrations of silver nanoparticles (20 nm) and magnesium oxide nanoparticles (50 nm) were included in the PCR mixture to improve the sensitivity of PCR for the detection of tomato leaf curl virus. The inclusion of nanoparticles in single or combination in PCR mixture has resulted in improvement of PCR sensitivity. Four-fold improvement was exhibited by the inclusion of 3 ng/µL silver nanoparticles, whereas the combination of silver and magnesium oxide nanoparticles (3 ng/µL and 200 ng/µL, respectively), resulted in a 4.5-fold improvement. The inclusion of 200 ng/µL of magnesium oxide nanoparticles in the PCR mixture exhibited a 7.6-fold increase in PCR sensitivity. Replacement of magnesium chloride with a combination of silver and magnesium oxide nanoparticles (3 ng/µL and 275 ng/µL, respectively) resulted in a 12-fold increase. A 13-fold improvement in PCR sensitivity was observed by the replacement of magnesium chloride in PCR buffer with 275 ng/µL of magnesium oxide nanoparticles. This could also produce detectable amplicon in PCR with a minimum of 25 cycles, resulting in a 26.5% reduction in the duration of PCR. This is the first report on the use of magnesium oxide nanoparticles in PCR for the early detection and better management of tomato leaf curl virus. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03842-2.

The establishment and application of a dual Nano-PCR detection method for feline calicivirus and feline herpesvirus type I.

Feline calicivirus (FCV) and Feline herpesvirus type I (FHV-I) are the main pathogens causing upper respiratory tract infections in cats, and some wild animals. These two viruses always coinfection and cause serious harm to pet industry and wild animals protection. Established a rapid and accurate differential diagnosis method is crucial for prevention and control of disease, however, the current main detection method for these two viruses, either is low sensitivity (immunochromatographic strip), or is time-consuming and cannot differential diagnosis (conventional single PCR). Nanoparticle-assisted polymerase chain reaction (Nano-PCR) is a recently developed technique for rapid detection method of virus and bacteria. In this study, we described a dual Nano-PCR assay through combining the nanotechnology and PCR technology, which for the clinical simultaneous detection of FCV and FHV-I and differential diagnosis of upper respiratory tract infections in cats or other animals. Under optimized conditions, the optimal annealing temperature for dual Nano-PCR was 51.5°C, and specificity test results showed it had no cross reactivity to related virus, such as feline panleukopenia virus (FPV), feline Infectious peritonitis virus (FIPV) and rabies virus (RABV). Furthermore, the detection limit of dual Nano-PCR for FCV and FHV-I both were 1 × 10-8 ng/µL, convert to number of copies of virus DNA was 6.22 × 103copies/µL (FCV) and 2.81 × 103copies/µL (FHV-I), respectively. The dual Nano-PCR detected result of 52 cat clinical samples, including ocular, nasal and faecal swabs, and (3 FCV-positive samples), was consistent with ordinary PCR and the clinical detection results. The dual Nano-PCR method established in this study with strong specificity and high sensitivity can be used for virus nucleic acid (FCV and FHV-I) detection of clinical samples of feline upper respiratory tract infections feline calicivirus and feline herpesvirus while providing support for the early diagnosis of cats that infected by FCV and FHV-I.

Establishment and preliminary application of nanoparticle-assisted PCR assay for detection of Cryptosporidium spp.

Cryptosporidium is an important intestinal protozoan parasite that causes diarrhoea in humans and animals. To rapidly and specifically detect Cryptosporidium spp., we designed a pair of primers based on the small subunit ribosomal RNA (SSU rRNA) gene of Cryptosporidium spp. to be used in a new nanoparticle-assisted PCR (nano-PCR) assay. The minimum detectable concentration (1.02 pg) of this nano-PCR was 10 times more sensitive than conventional PCR using the same primer pair. The DNA samples of C. parvum, C. baileyi, C. xiaoi, C. ryanae, and C. andersoni were successfully detected by the nano-PCR. No amplifications were evident with DNA samples of some common intestinal pathogens, including Eimeria tenella, Blastocystis sp., Giardia lamblia, Enterocytozoon bieneusi, and Balantidium coli. To validate the clinical usefulness of the novel nano-PCR, a total of 40 faecal samples from goats, camels, calves, and chickens were examined. The positive rate of Cryptosporidium spp. was 27.5% (11/40), which was consistent with that of an established nested PCR. These results indicate that the novel nano-PCR assay enables the rapid, specific, and accurate detection of Cryptosporidium infection in animals. The findings provide a technical basis for the clinical diagnosis, prevention, and control of cryptosporidiosis.

Phylogenetic analysis of porcine circovirus type 3 and porcine circovirus type 2 in China detected by duplex nanoparticle-assisted PCR.

Porcine circovirus 2 causes different clinical syndromes resulting in a significant economic loss in the pork industry and PCV3 infection was mainly distributed in the pig herds exhibiting PDNS and reproductive failure symptoms and was widely prevalent in the swine population in several states of the USA. Subsequently, PCV3 was also detected in the Chinese pig herds in Hubei and Shandong Provinces, etc. In a screening of 265 clinical samples from Beijing, Zhejiang, Guangdong and Hebei in China for PCV2, PCV3 and co-infection of PCV2 and PCV3, 14.2%, 16.6% and 6.8% of the samples tested positive, respectively. Under novel and optimized nano-dPCR reaction conditions, two specific fragments of 528 bp (PCV2) and 251 bp (PCV3) were amplified, respectively. No fragments were amplified when other porcine viruses were used as template. In this study, three PCV2 and three PCV3 isolates were evaluated by phylogenetic and alignment analysis and the results revealed that the PCV2 and PCV3 isolates appear new nucleotide substitutions and PCV3 isolates from China still included branch of porcine circovirus in USA.

Development of a nano-particle-assisted PCR assay for detection of duck tembusu virus.

UNLABELLED: Duck tembusu virus (DTMUV) has caused significant economic losses to the poultry industry in China since the spring of 2010. In this study, a nano-PCR assay targeting E gene of DTMUV was developed and their sensitivities and specificities were investigated. Under the optimized conditions of nano-PCR assay for detection of DTMUV, the nano-PCR assay was 10-fold more sensitive than a conventional PCR assay. The lower detection limit of the nano-PCR assay was 1·8 × 10(2)  copies µl(-1) of DTMUV RNA, as no cross-reaction was observed with other viruses. This is the first report to demonstrate the application of a nano-PCR assay for the detection of DTMUV. The sensitive, and specific nano-PCR assay developed in this study can be applied widely in clinical diagnosis and field surveillance of DTMUV-infection. SIGNIFICANCE AND IMPACT OF THE STUDY: A nanoparticle-assisted polymerase chain reaction (nano-PCR) assay was developed in this study for the rapid detection of duck tembusu virus (DTMUV) with high sensitivity and specificity. This technique has potential application in both clinical diagnosis and field surveillance of DTMUV-infection.