Development of SYBR green-based real-time PCR and duplex nested PCR assays for quantitation and differential detection of species- or type-specific porcine Torque teno viruses.
ABSTRACT Porcine Torque teno virus (TTV), a single-stranded circular DNA virus, has been incriminated in swine diseases recently. Multiple infection with porcine TTV species 1 (PTTV1) and species 2 (PTTV2), each consisting of two types (PTTV1a and 1b) or subtypes (PTTV2b and 2c), in a single pig had been reported by our group previously. The present study described three novel assays for quantitation and differential detection of porcine TTV. First, we developed two SYBR green-based real-time PCR assays to quantify viral loads of two porcine TTV species, respectively. The PTTV1- and PTTV2-specific real-time PCR primer sequences were selected to target conserved regions identified by multiple alignments of ten available porcine TTV full-length genomes. Furthermore, by coupling the two singleplex PCR assays, a duplex real-time PCR assay followed by melting curve analysis was established for simultaneous detection and differentiation of PTTV1 and PTTV2. In addition, a type-specific duplex nested PCR was also developed to simultaneously detect and distinguish between the two types, PTTV1a and 1b, in PTTV1 species. These assays provide rapid and practical tools for molecular diagnosis of species- or type-specific porcine TTV.
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ABSTRACT: BACKGROUND: Quantification of titers of ubiquitous viruses such as Torque teno virus (TTV) that do not cause clinical symptoms might be helpful in assessing the immune status of an individual. We hereby describe the validation of a SYBR Green-based TTV quantification method for plasma samples. METHODS: Plasmids with TTV specific inserts were used for preparing standards and absolute quantification of TTV was performed using SYBR Green methodology. The method was assessed for its accuracy and precision (intra and inter-day) on four non-consecutive days. TTV was also quantified from plasma samples of 20 healthy volunteers and from 30 hematopoietic stem cell transplant (HSCT) recipients. RESULTS: The assay was specific and showed satisfactory efficiency (82.2 %, R2=0.99) with the limit of quantification defined as 100 copies per reaction. The assay had good precision (inter and intra-day coefficient of variation in cycle threshold (CT) < 4%) and accuracy (100 +/- 10 %) in the range of 100 to 1010 copies/reaction. We found TTV loads ranging from 2.5 -- 4.07 log copies/mL of plasma with CT (mean +/- SD) of 33.8 +/- 1.77 in healthy individuals and 2.06 -- 8.49 log copies/mL of plasma with CT (mean +/- SD) of 24.3 +/- 1.04 in HSCT recipients. CONCLUSION: SYBR Green-based q-PCR assay combines simplicity with satisfactory sensitivity and may be suitable for monitoring the immune status of transplant recipients, where TTV loads over time may serve as a marker for immune reconstitution in human plasma samples.Virology Journal 06/2013; 10(1):191. · 2.09 Impact Factor
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ABSTRACT: Torque teno viruses (TTVs) are a large group of vertebrate-infecting small viruses with circular single-stranded DNA, classified in the Anelloviridae family. In swine, two genetically distinct species, Torque teno sus virus 1a (TTSuV1a) and 1b (TTSuV1b) are currently grouped into the genus Iotatorquevirus. More recently, a novel Torque teno sus virus species, named Torque teno sus virus k2b (TTSuVk2b), has been included with Torque teno sus virus k2a (TTSuVk2a) into the genus Kappatorquevirus. In the present study, TTSuV1 (TTSuV1a and TTSuV1b), TTSuVk2a and TTSuVk2b prevalence was evaluated in 721 serum samples of healthy pigs from Sardinian farms, insular Italy. This is the largest study to date on the presence of TTSuV in healthy pigs in Italy. The global prevalence of infection was 83.2% (600/721), being 62.3% (449/721), 60.6% (437/721), and 11.5% (83/721) the prevalence of TTSuV1, TTSuVk2a and TTSuVk2b, respectively. The rate of co-infection with two and/or three species was also calculated, and data show that co-infections were significantly more frequent than infections with single species, and that TTSuV1+TTSuVk2a double infection was the prevalent combination (35.4%). Quantitative results obtained using species-specific real time-qPCR evidenced the highest mean levels of viremia in the TTSuV1 subgroup, and the lowest in the TTSuVk2b subgroup. Interestingly, multiple infections with distinct TTSuV species seemed to significantly affect the DNA load and specifically, data highlighted that double infection with TTSuVk2a increased the viral titers of TTSuV1, likewise the co-infection with TTSuVk2b increased the titers of TTSuVk2a.PLoS ONE 01/2014; 9(11):e113720. · 3.53 Impact Factor
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ABSTRACT: BACKGROUND: Torque teno sus viruses (TTSuVs) are non-enveloped viruses and have single-stranded, negative sense circular DNA genomes and are widely distributed in pigs. But till now, the prevalence of TTSuVs with porcine circovirus type 2 (PCV2) in pig herds of China is not very clear; and the genetic variation among different TTSuVs isolate is very large and need to divide the subgroups. In this study, the co-infection with TTSuVs and porcine circovrius (PCV) in the pig population of China was investigated and the subgroups of all TTSuVs genomes in Genbank were divided. RESULTS: Results showed that the rate of co-infection with TTSuV1 and TTSuV2 reached 75% in PCV2-positive samples. Also Two TTSuV1 and four TTSuV2 isolates genome sequences were obtained, and the similarity of all TTSuV1 and TTSuV2 genomic sequences in GenBank were compared. Phylogenetic trees indicated that both the TTSuV1 and TTSuV2 sequences could be divided into four genotypes. Interestingly, the sub-genotypes TTSuV1d, TTSuV2c and TTSuV2d exist only in the pig population of China. CONCLUSIONS: This study demonstrates that co-infection with TTSuVs and PCVs is very common in the pig population of China, in which the viruses maybe contribute to clinical diseases cooperatively. In addition, three new subgroups of TTSuVs emerged in China for the first time and a high level of variation among different isolates of TTSuV1 and TTSuV2 was indicated by their genetic diversity.Virology Journal 06/2013; 10(1):189. · 2.09 Impact Factor