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Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.

Huang Y, Xing N, Wang Z, Zhang X, Zhao X, Du Q, Chang L, Tong D - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, P. R. China.

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.

No MeSH data available.


Related in: MedlinePlus

Comparison of different probes-functionalized MMPs and oligos-functionalized AuNPs.(A) Functional magnetic microparticles MMP-p1, -p2, -p3, -p4, -p5 and -p6 were incubated with RNA of TGEV in hybridization buffer at 40°C for 30 minutes, followed by washing and magnetic separation. Then the MMP-RNA complexes were reverse transcribed into cDNA, which were detected by TGEV-specific RT-PCR. M: Trans 2K Plus DNA Marker; 1: MMP1; 2: MMP2; 3: MMP3; 4: MMP4; 5: MMP5; 6: MMP6. (B) TGEV RNA was incubated with oligo1, oligo2, oligo3, oligo4, oligo5 or oligo6 functionalized Au-NPs at 50°C for 40 min. Then the complexes were washed and precipitated by centrifugation, followed by reverse transcription and TGEV specific RT-PCR detection. M: Trans 2K Plus DNA Marker; 1: oligo1; 2: oligo2; 3: oligo3; 4: oligo4; 5: oligo5; 6: oligo6.
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pone.0141545.g002: Comparison of different probes-functionalized MMPs and oligos-functionalized AuNPs.(A) Functional magnetic microparticles MMP-p1, -p2, -p3, -p4, -p5 and -p6 were incubated with RNA of TGEV in hybridization buffer at 40°C for 30 minutes, followed by washing and magnetic separation. Then the MMP-RNA complexes were reverse transcribed into cDNA, which were detected by TGEV-specific RT-PCR. M: Trans 2K Plus DNA Marker; 1: MMP1; 2: MMP2; 3: MMP3; 4: MMP4; 5: MMP5; 6: MMP6. (B) TGEV RNA was incubated with oligo1, oligo2, oligo3, oligo4, oligo5 or oligo6 functionalized Au-NPs at 50°C for 40 min. Then the complexes were washed and precipitated by centrifugation, followed by reverse transcription and TGEV specific RT-PCR detection. M: Trans 2K Plus DNA Marker; 1: oligo1; 2: oligo2; 3: oligo3; 4: oligo4; 5: oligo5; 6: oligo6.

Mentions: TGEV, an enveloped virus of the coronaviridae family, contains a single-stranded positive-sense RNA genome of 28.5 kb. 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 (gRNA) and subgenomic mRNAs (sgmRNAs) to encode structural and nonstructural proteins of virus [21, 29, 30]. In this assay, we first quantified viral number using primers targeted to TGEV gRNA and different sgmRNAs and found that viral numbers were significantly different when the primers were targeted to TGEV gRNA and different sgmRNAs (data not shown). Therefore, we designed probes targeted to replicase protein-encoding region ORF1a to quantify the amount of TGEV gRNA. Six targeted regions were selected from the 5’ end, middle and 3’ end of the ORF1a for designing probes 1 to 6 (Table 1). All the targeted sequences of these probes are highly conserved in variety of TGEV strains. Probes 1 to 6 were coated to magnetic microparticles to prepare functional magnetic beads MMP-p1, -p2, -p3, -p4, -p5 and -p6, respectively. To select the optimal probes for capturing of TGEV genomic RNA, capture efficiency of these designed probes were determined by conventional RT-PCR assay. The results showed that all six probes could capture TGEV RNA, however MMP-p1 and MMP-p2 exhibited higher capture ability for TGEV RNA (Fig 2A). Next, we assessed capture efficiency of functionalized gold nanoparticles coated with oligonucleotides (oligo) 1 to 6 that shared same targeted sequence with probe 1 to 6. The prepared functionalized Au-NPs were precipitated by centrifugation and were detected by RT-PCR assay. The results showed that oligo 1 and 2-coated Au-NP possessed higher binding ability with TGEV nucleic acid (Fig 2B). Therefore, in the UNDP-PCR assay for TGEV, probe 1-functionalized MMPs and oligo 2-functionalized Au-NPs are optimal for capture of TGEV RNA and formation of sandwich complex.


Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.

Huang Y, Xing N, Wang Z, Zhang X, Zhao X, Du Q, Chang L, Tong D - PLoS ONE (2015)

Comparison of different probes-functionalized MMPs and oligos-functionalized AuNPs.(A) Functional magnetic microparticles MMP-p1, -p2, -p3, -p4, -p5 and -p6 were incubated with RNA of TGEV in hybridization buffer at 40°C for 30 minutes, followed by washing and magnetic separation. Then the MMP-RNA complexes were reverse transcribed into cDNA, which were detected by TGEV-specific RT-PCR. M: Trans 2K Plus DNA Marker; 1: MMP1; 2: MMP2; 3: MMP3; 4: MMP4; 5: MMP5; 6: MMP6. (B) TGEV RNA was incubated with oligo1, oligo2, oligo3, oligo4, oligo5 or oligo6 functionalized Au-NPs at 50°C for 40 min. Then the complexes were washed and precipitated by centrifugation, followed by reverse transcription and TGEV specific RT-PCR detection. M: Trans 2K Plus DNA Marker; 1: oligo1; 2: oligo2; 3: oligo3; 4: oligo4; 5: oligo5; 6: oligo6.
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pone.0141545.g002: Comparison of different probes-functionalized MMPs and oligos-functionalized AuNPs.(A) Functional magnetic microparticles MMP-p1, -p2, -p3, -p4, -p5 and -p6 were incubated with RNA of TGEV in hybridization buffer at 40°C for 30 minutes, followed by washing and magnetic separation. Then the MMP-RNA complexes were reverse transcribed into cDNA, which were detected by TGEV-specific RT-PCR. M: Trans 2K Plus DNA Marker; 1: MMP1; 2: MMP2; 3: MMP3; 4: MMP4; 5: MMP5; 6: MMP6. (B) TGEV RNA was incubated with oligo1, oligo2, oligo3, oligo4, oligo5 or oligo6 functionalized Au-NPs at 50°C for 40 min. Then the complexes were washed and precipitated by centrifugation, followed by reverse transcription and TGEV specific RT-PCR detection. M: Trans 2K Plus DNA Marker; 1: oligo1; 2: oligo2; 3: oligo3; 4: oligo4; 5: oligo5; 6: oligo6.
Mentions: TGEV, an enveloped virus of the coronaviridae family, contains a single-stranded positive-sense RNA genome of 28.5 kb. 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 (gRNA) and subgenomic mRNAs (sgmRNAs) to encode structural and nonstructural proteins of virus [21, 29, 30]. In this assay, we first quantified viral number using primers targeted to TGEV gRNA and different sgmRNAs and found that viral numbers were significantly different when the primers were targeted to TGEV gRNA and different sgmRNAs (data not shown). Therefore, we designed probes targeted to replicase protein-encoding region ORF1a to quantify the amount of TGEV gRNA. Six targeted regions were selected from the 5’ end, middle and 3’ end of the ORF1a for designing probes 1 to 6 (Table 1). All the targeted sequences of these probes are highly conserved in variety of TGEV strains. Probes 1 to 6 were coated to magnetic microparticles to prepare functional magnetic beads MMP-p1, -p2, -p3, -p4, -p5 and -p6, respectively. To select the optimal probes for capturing of TGEV genomic RNA, capture efficiency of these designed probes were determined by conventional RT-PCR assay. The results showed that all six probes could capture TGEV RNA, however MMP-p1 and MMP-p2 exhibited higher capture ability for TGEV RNA (Fig 2A). Next, we assessed capture efficiency of functionalized gold nanoparticles coated with oligonucleotides (oligo) 1 to 6 that shared same targeted sequence with probe 1 to 6. The prepared functionalized Au-NPs were precipitated by centrifugation and were detected by RT-PCR assay. The results showed that oligo 1 and 2-coated Au-NP possessed higher binding ability with TGEV nucleic acid (Fig 2B). Therefore, in the UNDP-PCR assay for TGEV, probe 1-functionalized MMPs and oligo 2-functionalized Au-NPs are optimal for capture of TGEV RNA and formation of sandwich complex.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, P. R. China.

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.

No MeSH data available.


Related in: MedlinePlus