SYBR Green I-based Quantitative PCR Research Articles

Development of SYBR Green I-based quantitative PCR assay for identification of porcine circovirus 1, 2 and 3

Porcine Circovirus (PCV) includes Porcine Circovirus 1(PCV1), Porcine Circovirus 2 (PCV2) and Porcine Circovirus 3 (PCV3). In recent years, co-infection exists between PCV1, PCV2 and PCV3 serotypes. Therefore, it is particularly necessary to establish a fast, specific and sensitive SYBR Green I real-time quantitative PCR detection method for PCV1, PCV2 and PCV3. In this experiment, specific primers were selected and the reaction conditions were optimized. A real-time quantitative PCR identification method was established. The results showed the detection limits of this assay were 40.3 copies/μl for PCV1, 25.2 copies/μl for PCV2 and22.4 copies/ μl for PCV3. There was no cross-reactivity with swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine pseudorabies virus (PRV) and porcine parvovirus (PPV). The intra-assay and inter-assay coefficients of variation were less than 1%. The test results of 100 PCV suspected positive samples revealed that the PCV1, PCV2 and PCV3 singular infection rate was 10% (10/100), 64% (64/100) and 52% (52/100), respectively. The PCV1 and PCV2 co-infection rate was 8% (8/100), the PCV1 and PCV3 co-infection rate was 7% (7/100), the PCV2 and PCV3 co-infection rate was 26% (26/100), and the PCV1, PCV2 and PCV3 co-infection rate was 7% (7/100). This method has good specificity, sensitivity and stability. It provides a promising tool for rapid differential detection of PCV1, PCV2 and PCV3.

Detection and genetic characteristics of porcine bocavirus in central China.

To investigate the epidemic profile and genetic diversity of porcine bocavirus (PBoV), 281 clinical samples, including 236intestinal tissue samples and 45 fecal samples were collected from diarrheic piglets on 37 different pig farms in central China, and two SYBR Green I-based quantitative PCR assays were developed to detect PBoV1/2 and PBoV3/4/5, respectively. One hundred forty-eight (52.67%) of the 281 clinical samples were positive for PBoV1/2, 117 (41.63%) were positive for PBoV3/4/5, 55 (19.57%) were positive for both PBoV1/2 and PBoV3/4/5, and 86.49% (32/37) of the pig farms were positive for PBoV. Overall, the prevalence of PBoV was 74.73% (210/281) in central China. Subsequently, nearly full-length genomic sequences of two PBoV strains (designated CH/HNZM and PBoV-TY) from two different farms were determined. Phylogenetic analysis demonstrated that the two PBoV strains obtained in this study belonged to the PBoV G2 group and had a close relationship to 10 other PBoV G2 strains but differed genetically from PBoV G1, PBoV G3, and seven other bocaviruses. CH/HNZM and PBoV-TY were closely related to the PBoV strain GD18 (KJ755666), which may be derived from the PBoV strains 0912/2012 (MH558677) and 57AT-HU (KF206160) through recombination. Compared with reference strain ZJD (HM053694)-China, more amino acid variation was found in the NS1 proteins of CH/HNZM and PBoV-TY. These data extend our understanding of the molecular epidemiology and evolution of PBoV.

The development of a rapid SYBR Green I-based quantitative PCR for detection of Duck circovirus

This report describes a one-step real-time polymerase chain reaction assay based on SYBR Green I for detection of a broad range of duck circovirus (DuCV). Align with all DuCV complete genome sequences and other Genus Circovirus download from the GenBank (such as goose circovirus, pigeon circovirus), the primers targets to the replicate gene of DuCV were designed. The detection assay was linear in the range of 1.31 × 102-1.31 × 107 copies/μL. The reaction efficiency of the assay using the slope (the slope was -3.349) and the Y-intercept was 37.01 from the linear equation was estimated to be 0.99 and the correlation coefficient (R2) was 0.993. A series of experiments were carried out to assess the reproducibility, sensitivity, and specificity of the assay, following by the low intra-assay and inter-assay CVs for CT values obtained with the standard plasmids. The intra-assay CVs were equal or less than 1.89% and the inter-assay CVs were equal or less than 1.26%. There was no cross-reaction occurred with nucleic acids extracted from RA (Riemerella anatipestifer), E. coli (Escherichia coli), Duck Cholera (Pasteurella multocida), Avian influenza virus, avian paramyxovirus, Muscovy duck parvovirus, Duck reovirus, Duck hepatitis A virus as control templates. The nucleic acids extracted from samples of healthy ducks were used as negative controls. The assay was specific and reproducible. The established real time PCR was used to detect 45 DuCV-negative samples, which were tested using conventional PCR under the developed optimal conditions, each 15 for embryonated eggs, non-embryonated budgerigar eggs, newly hatched duck, the mixture of the lung, liver, spleen which were analysis for the presence of DuCV DNA, to conform that whether the DuCV can be transmitted vertically. Meanwhile, no positive result was shown by the real-time PCR method. The SYBR Green I-based quantitative PCR can therefore be practically used as an alternative diagnostic tool and a screening method for ducks infected with duck circovirus.

Using the Taguchi method for rapid quantitative PCR optimization with SYBR Green I

Here, we applied the Taguchi method, an engineering optimization process, to successfully determine the optimal conditions for three SYBR Green I-based quantitative PCR assays. This method balanced the effects of all factors and their associated levels by using an orthogonal array rather than a factorial array. Instead of running 27 experiments with the conventional factorial method, the Taguchi method achieved the same optimal conditions using only nine experiments, saving valuable resources.

Molecular Characterization and SYBR Green I-Based Quantitative PCR for Duck Hepatitis Virus Type 1

To determine the genomic sequence of a duck hepatitis virus type 1 (DHV-1) strain, real-time quantitative polymerase chain reaction (RTQ-PCR) assay based on SYBR Green I technology was developed to target 3D gene of DHV-1. Comparative sequence analysis showed that the genome has a typical picornarivus genetic organization, and strain DHV-1 R genetic organaiztion is 5′ untranslated region (UTR)-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3′ UTR, DHV-1 R has close relationship with Parechovirus, and has 95.1-99.1% nucleotide sequence identity with other DHV-1 strains. Based on the DHV-1 sequences in GenBank, three pairs of specific primers were designed to amplify DHV-1 using real-time PCR. The results showed that real-time PCR Tm value is 85.6°C and the real-time PCR provides a broad dynamic range, detecting from 10 2 to 10 9 copies of DHV-1 cDNA per reaction. No cross-reactions were found in specimens containing DPV, AIV and NDV. It is concluded that DHV-1 belongs to a new group of the family Picornaviridae that may form a separate genus most closely related to the genus Parechovirus. All results showed that the real-time PCR has high sensitivity and specificity to detect DHV-1 using SYBR Green I dissociation curve analysis, isolates can be distinguished by their melting temperature. These methods are rapid, sensitive, and reliable, and can be readily adapted for detection of DHV-1 from other clinical samples.

Detection of PCV2 DNA by SYBR Green I-based quantitative PCR

We developed an assay for the detection and quantitation of porcine circovirus type 2 (PCV2) with the SYBR Green I-based real-time PCR. The real-time PCR provides a broad dynamic range, detecting from 10(3) to 10(11) copies of DNA per reaction. No cross-reactions were found in specimens containing PCV1. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, real-time PCR can be used as a routine assay for the clinical diagnosis of PCV2 infection. In this study we applied real-time PCR assay to 80 clinical samples, collected from 40 pigs with postweaning multisystemic wasting syndrome (PMWS) and 40 healthy pigs in comparison with conventional PCR assay. In 56 of 80 samples, PCV2 DNA was detected by conventional PCR assay. All samples positive for PCV2 DNA in conventional PCR assay were also positive in real-time assay, and 12 of 24 samples that tested negative for PCV2 DNA in the conventional assay were tested positive in real-time PCR assay. Real-time PCR assay increased the number of samples in which PCV2 was detected by 15%. It is, therefore, considered to be a useful tool for the detection of PCV2.