TY - JOUR
T1 - Multiplex SYBR Green and duplex TaqMan real-time PCR assays for the detection of Photorhabdus Insect-Related (Pir) toxin genes pirA and pirB
AU - Cruz-Flores, Roberto
AU - Mai, Hung Nam
AU - Dhar, Arun K.
N1 - Funding Information:
Funding for this research was provided by the College of Agriculture & Life Sciences in The University of Arizona to Arun K. Dhar. This work was also partially supported by the USDA National Institute of Food and Agriculture , Animal Health project 1006512 . The authors would like to thank Brenda Noble and Paul Schofield for performing an AHPND laboratory challenge and generating P. vannamei tissues.
Funding Information:
Funding for this research was provided by the College of Agriculture & Life Sciences in The University of Arizona to Arun K. Dhar. This work was also partially supported by the USDA National Institute of Food and Agriculture, Animal Health project 1006512. The authors would like to thank Brenda Noble and Paul Schofield for performing an AHPND laboratory challenge and generating P. vannamei tissues.
Publisher Copyright:
© 2018
PY - 2019/2
Y1 - 2019/2
N2 - 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.
AB - 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.
KW - Acute hepatopancreatic necrosis disease
KW - Early mortality syndrome
KW - Multiplex real-time PCR
KW - Pathogen detection
KW - SYBR Green
KW - TaqMan
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U2 - 10.1016/j.mcp.2018.12.004
DO - 10.1016/j.mcp.2018.12.004
M3 - Article
C2 - 30576786
AN - SCOPUS:85058812496
SN - 0890-8508
VL - 43
SP - 20
EP - 28
JO - Molecular and Cellular Probes
JF - Molecular and Cellular Probes
ER -