Quantitative assay for measuring the Taura syndrome virus and yellow head virus load in shrimp by real-time RT-PCR using SYBR Green chemistry

Abstract

Taura syndrome virus (TSV) and yellow head virus (YHV) are the two RNA viruses infecting penaeid shrimp (Penaeus sp.) that have caused major economic losses to shrimp aquaculture. A rapid and highly sensitive detection and quantification method for TSV and YHV was developed using the GeneAmp 5700 Sequence Detection System and SYBR Green chemistry. The reverse transcriptase polymerase chain reaction (RT-PCR) mixture contained a fluorescent dye, SYBR Green, which exhibits fluorescence enhancement upon binding to double strand cDNA. The enhancement of fluorescence was found to be proportional to the initial concentration of the template cDNA. A linear relationship was observed between input plasmid DNA and cycle threshold (C(T)) values for 10(6) down to a single copy of both viruses. To control for the variation in sample processing and in reverse transcription reaction among samples, shrimp beta-actin and elongation factor-1alpha (EF-1alpha) genes were amplified in parallel with the viral cDNA. The sensitivity and the efficiency of amplification of EF-1alpha was greater than beta-actin when compared to TSV and YHV amplification efficiency suggesting that EF-1alpha is a better internal control for the RT-PCR detection of TSV and YHV. In addition, sample to sample variation in EF-1alpha C(T) value was lower than the variation in beta-actin C(T) value of the corresponding samples. The specificity of TSV, YHV, EF-1alpha and beta-actin amplifications was confirmed by analyzing the dissociation curves of the target amplicon. The C(T) values of TSV and YHV samples were normalized against EF-1alpha C(T) values for determining the absolute copy number from the standard curve of the corresponding virus. The method described here is highly robust and is amenable to high throughput assays making it a useful tool for diagnostic, epidemiological and genetic studies in shrimp aquaculture.

Fig. 1

The standard curve for TSV (A) and YHV (B) obtained by SYBR Green PCR using plasmid DNA as template. The number of copies of TSV plasmid DNA added to each reaction mixture (corresponding to the numbers on the linear curve in panel A) were as follows: (1) 2.42×10⁶, (2) 2.42×10⁵, (3) 2.42×10⁴, (4) 2.42×10³, (5) 2.42×10², (6) 2.42×10¹, (7) 12.1, (8) 6.05, (9) 3.03 and (10) 1.51. For YHV sample, the plasmid copy numbers (corresponding to the numbers on the linear curve in panel B) were as follows: (1) 2.12×10⁶, (2) 2.12×10⁵, (3) 2.12×10⁴, (4) 2.12×10³, (5) 2.12×10², (6) 2.12×10¹, (7) 10.6, (8) 5.3, (9) 2.6 and (10) 1.33.

Fig. 2

Relative amplification efficiency curves for (A) TSV and (B) YHV compared to EF-1α and β-actin using cDNA as template.

Fig. 3

The amplification plots and the corresponding dissociation curves of TSV, EF-1α and β-actin genes. The melting temperature (T_m) of each amplicon is shown alongside its dissociation curve.

Fig. 4

The amplification plots and the dissociation curves of YHV, EF-1α and β-actin genes. The melting temperature (T_m) of each amplicon is shown alongside its dissociation curve.