Abstract
Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.
Highlights
Along with the development of large-scale and intensive swine production, mixed infections of multiple pathogens are increasingly becoming common in swine farms
The first two-thirds of the viral genome encodes two replicases and only exists in genomic RNA, while the last third exists in both genomic RNAs and subgenomic mRNAs to encode structural and nonstructural proteins of virus [21, 29, 30]
We first quantified viral number using primers targeted to transmissible gastroenteritis virus (TGEV) genomic RNAs (gRNA) and different subgenomic mRNAs (sgmRNAs) and found that viral numbers were significantly different when the primers were targeted to TGEV gRNA and different sgmRNAs
Summary
Along with the development of large-scale and intensive swine production, mixed infections of multiple pathogens are increasingly becoming common in swine farms. It is imperative to establish an effective and rapid method to detect multiple DNA and RNA viruses simultaneously in single sample. Traditional diagnostic methods of DNA and RNA viruses are mainly dependent on detection of viral proteins and nucleic acids. LAMP and real-time PCR are more sensitive than conventional PCR or RT-PCR, LAMP can only detect one pathogen at a time and LAMP products are difficult to identify, while real-time PCR requires special or expensive instruments and shows false positive results [17, 18]. RNA is degradable as compared to DNA, so in PCRbased methods of detecting RNA viruses, viral genomic RNA extracted from field samples should be utilized to synthesize complementary DNA (cDNA) first, which are time-consuming and labor-intensive
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