Multiplex SYBR Green and duplex TaqMan real-time PCR assays for the detection of Photorhabdus Insect-Related (Pir) toxin genes pirA and pirB

Abstract

Acute hepatopancreatic necrosis disease (AHPND), also known as Early mortality syndrome (EMS), is a recently emerged lethal disease that has caused major economic losses in shrimp aquaculture. The etiologic agents are Vibrio spp. that carry Photorhabdus Insect-Related (Pir) toxin genes pirA and pirB. A multiplex SYBR Green real-time PCR was developed that detects pirA, pirB, and two internal control genes, the shrimp 18S rRNA and the bacterial 16S rRNA genes in a single reaction. The pirB primers amplify the 3'-end of the pirB gene allowing the detection of Vibrio spp. mutants that contain a complete deletion of pirA and the partial deletion of pirB. The assay also detects mutants that contain the entire pirA gene and the deletion of the pirB gene. Since both toxin genes are needed for disease development, this assays can distinguish between pathogenic strains of Vibrio spp. that cause AHPND in shrimp and mutants that do not cause disease. The amplicons for pirA, pirB, 18S rRNA and 16S rRNA showed easily distinguishable melting temperatures of 78.21 ± 0.18, 75.20 ± 0.20, 82.28 ± 0.34 and 85.41 ± 0.21 °C respectively. Additionally, a duplex real-time PCR assay was carried out by designing TaqMan probes for the pirA and pirB primers. The diagnostic sensitivity and specificity was compared between the SYBR Green and TaqMan assays. Both assays showed similar sensitivity with a limit of detection being 10 copies for pirA and pirB, and neither assays showed any cross reaction with other known bacterial and viral pathogens in shrimp. The high sensitivity of both assays make them suitable for the detection of low copies of the pirA and pirB genes in AHPND causing Vibrio spp. as well as for detecting non-pathogenic mutants.

Keywords: Acute hepatopancreatic necrosis disease; Early mortality syndrome; Multiplex real-time PCR; Pathogen detection; SYBR Green; TaqMan.

Graphical abstract

Fig. 1

The pirA and pirB primer locations on the virulence plasmid in the reference strain Vibrio parahaemolyticus A3. The primers pirA F1, pirA R1, pirB F1 and pirB R1 for dual detection of the pirA and pirB genes are shown in blue. The primers pirA F2, pirA R2, pirB F2 and pirB R2 for dual detection of the pirA and pirB genes are shown in green. The primers pirA F3, pirA R3, pirB F3 and pirB R3 for dual detection of the pirA and pirB genes are shown in red. The pirB primers are located near the 3′ end of the pirB gene allowing the detection of mutants with a partial deletion of pirB.

Fig. 2

Melt curve analysis of PCR amplicons derived from AHPND-infected shrimp tissue and Vibrio spp. (A) Melt curve analysis of infected shrimp tissue for pirA, pirB, 18S rRNA and 16S rRNA, the amplicons presented melting temperatures of 78.21 ± 0.18, 75.20 ± 0.20, 82.28 ± 0.34 and 85.41 ± 0.21 °C, respectively. (B) Melt curve analysis of V. parahaemolyticus pirA negative strain. The melting temperatures of pirB and 16S rRNA are 75.20 ± 0.20 and 85.41 ± 0.21 °C respectively. Each amplicon shows a clearly defined peak. (C) Melt curve pattern of V. shiloi pirB negative strain. The melting temperatures of pirA and 16S rRNA are 78.21 ± 0.18 and 85.41 ± 0.21 °C respectively. In (D) we can observe the melt curve analysis of AHPND-causing strains of Vibrio spp. The melting temperatures of pirA, pirB and 16S rRNA are 78.21 ± 0.18, 75.20 ± 0.20 and 85.41 ± 0.21 °C respectively. In A, B, C and D each amplicon shows a clearly defined peak.

Fig. 3

Simultaneous amplification of the pirA and pirB genes by the duplex TaqMan assay. The amplification curves of pirA and pirB are indicated.

Fig. 4

Standard curve for the pirA and pirB genes. The VppirA80 and VppirB149 plasmids were serially diluted from 10⁶ to 10¹ copies/μl and were used as a template for PCR. The Ct values are plotted against the logarithm of their respective copy number. (A) A standard curve for the pirA gene generated using the SYBR Green assay with a log linear dynamic range of 6 orders of magnitude with an R² value of 0.998, slope of −3.34 and amplification efficiency of 99.12%. (B) A standard curve for the pirB gene generated using the SYBR Green assay with a log linear dynamic range of 6 orders of magnitude with an R² value of 0.999, slope of −3.381 and amplification efficiency of 97.59%. (C) A standard curve for the pirA gene generated using the TaqMan assay with a log linear dynamic range of 6 orders of magnitude with an R² value of 0.998, slope of −3.549 and amplification efficiency of 91.32%. (D) A standard curve for the pirB gene generated using the TaqMan assay with a log linear dynamic range of 6 orders of magnitude with an R² value of 0.980, slope of −3.101 and amplification efficiency of 110.14%.

Fig. 5

Detection sensitivity of the SYBR Green and TaqMan assays using AHPND-infected Penaeus vannamei from the experimental challenge test. (A) The SYBR Green assay was able to detect the pirA, pirB, 18S rRNA and 16S rRNA down to 200 fg of total DNA. (B) The TaqMan assay showed a similar sensitivity also detecting both genes down to 200 fg of total DNA. The red amplification plot represents 20 ng of total DNA, pirA (Ct = 19.39 ± 0.44), pirB (Ct = 17.11 ± 0.23). The yellow amplification plot represents 2 ng of total DNA pirA (Ct = 22.80 ± 0.41), pirB (Ct = 20.53 ± 0.24). The green amplification plot represents 200 pg of total DNA pirA (Ct = 26.49 ± 0.15), pirB (Ct = 23 ± 0.27). The turquoise amplification plot represents 20 pg of total DNA pirA (Ct = 29.34 ± 0.26), PirB (Ct = 27.19 ± 0.33). The light blue amplification plot represents 2 pg of total DNA pirA (Ct = 32.18 ± 12), pirB (Ct = 30.55 ± 0.27). The navy blue amplification plot represents 200 fg of total DNA pirA (Ct = 35.03 ± 0.32), pirB (Ct = 32.55 ± 0.30).