key: cord-0735722-32hlvdku
authors: Cruz-Flores, Roberto; Mai, Hung Nam; Dhar, Arun K.
title: Multiplex SYBR Green and duplex TaqMan real-time PCR assays for the detection of Photorhabdus Insect-Related (Pir) toxin genes pirA and pirB
date: 2018-12-18
journal: Mol Cell Probes
DOI: 10.1016/j.mcp.2018.12.004
sha: f2e653d48d4c3eb2197f86102e2b5f301b26af38
doc_id: 735722
cord_uid: 32hlvdku

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.

Acute hepatopancreatic necrosis disease (AHPND, initially referenced to as early mortality syndrome, EMS) is a deadly shrimp disease caused by particular Vibrio spp. [1] [2] [3] . The disease first emerged in China in 2009 and has rapidly spread throughout Southeast Asia to Vietnam, Malaysia, Thailand and reached Mexico in Latin America in 2013 [1, 4] . The impact of AHPND in shrimp farming at a global scale has been catastrophic with an estimated global loss of $1 billion per year [5] .

The etiologic agent of AHPND was shown to be a specific strain of Vibrio parahaemolyticus that carries the pirA and pirB genes homologous to the Pir (Photorhabdus insect-related) binary toxin of entomopathogenic bacteria [1, 3] . Since the initial report, several other Vibrio species including V. owensii [6] , V. campbelli [7] , V. harveyi [8] and Vibrio punensis [9] have been reported that cause AHPND. More recently, a non-Vibrio bacterium, Microccocus luteus, has been found that contains the pirA and pirB genes [10] .

The pathogenic Vibrio spp. harbor a large plasmid that ranges from 69 to 74 kb, on average of 33 copies per cell, and contains Photorhabdus Insect-Related (Pir) toxin genes pirA and pirB [3, 11, 12] . The binary toxin pirAB has been confirmed to be the etiological agent for AHPND [11] . To date, two conventional PCR based methods have been reported to detect both toxins genes pirA and pirB, a duplex conventional PCR reported by Han et al. [3] and a two-tube nested AP4 PCR developed by Dangtip et al. [13] . However, the two-tube nested AP4 PCR cannot detect deletion mutants that have only one gene pirA or pirB. A qPCR assay that detects pirA but does not detect mutants with the deletion of pirB has been reported [14] . The detection of both types of mutants is fundamental for the study of plasmid transmission dynamics and for recording the presence of the virulence plasmid since a recent study [2] suggests that the pirA and pirB genes may be lost or acquired by horizontal gene transfer via transposition or homologous recombination. In fact, the genome of a mutant strain of V. parahaemolyticus has been published that lacks the entire pirA gene and has a partial deletion of the 5′-end of the pirB gene [15] . In this study, we report a multiplex SYBR Green real-time PCR that detects the pirA and pirB toxin genes, and two internal control genes, the shrimp 18S rRNA and the bacterial 16S rRNA. Furthermore, we report a duplex TaqMan PCR that also detects pirA and pirB simultaneously. These assays will greatly aid in the detection and monitoring of low quantities of AHPND causing Vibrio spp. and mutant strains containing either pirA or pirB.

Three different primer pairs were designed with Geneious R11 [16] to detect the pirA and pirB toxin genes ( Table 1 ). The primers for the pirB were designed to amplify the 3'-end of the pirB gene allowing the detection of Vibrio spp. that have a partial deletion of this gene (Fig. 1) . The genes used as internal controls for shrimp and bacteria were β-Actin, Elongation factor1-alpha, Glyceroldehyde-3 phosphate dehydrogenase, 18S rRNA, and 16S rRNA ( Table 2 ).

Multiplex SYBR Green real-time PCR was performed using a StepOnePlus PCR system (Applied Biosystems ™). Each assay was carried out in a total volume of 20 μl containing 1 μl of template DNA, 10 μl of PowerUp™ SYBR™ Green Master Mix (2X), 125 nM of pirA primers (Set 1, Set 2 and Set3), 150 nM of pirB primers (Set 1, Set 2 and Set2), 75 nM of shrimp internal control primers (β-Actin, EF1-α, G3PD and 18S rRNA) and 350 nM of 16S rRNA bacterial internal control primers. The primer concentration was determined by testing each primer set with concentrations that ranged from 50 nM to 500 nM. Each primer set listed in Table 1 was tested with one shrimp internal control primer pair and the bacterial 16S rRNA internal control primers. The real-time PCR conditions consisted of a UDG activation at 50°C for 2 min, denature and Dual-Lock ™ DNA polymerase activation at 95°C for 2 min, followed by 40 cycles at 95°C for 3s and 59°C for 30s. Following amplification, the melt curve analysis was performed. The reaction temperature was increased to 95°C for 15s, then decreased to 60°C for 1 min, and increased to 95°C at a rate 0.1°C per second with a continuous fluorescence monitoring. The melt curves were used to determine if the primers were compatible and primer combinations that showed four clear specific amplification peaks were considered adequate for the multiplex real-time PCR.

TaqMan probes were designed for primer pair pirA F1/R1 and pirB F1/R1 using Geneious R11 [16] . The TaqMan probe for pirA F1/R1 (5′-GAACCAAACGGAGGCGTCA-3′) was synthesized and labeled with 6-Carboxy-4′,5′-Dichloro-2′, 7′-Dimethoxyfluorescein, Succinimidyl Ester (JOE) on the 5′ end and N, N, N′, N′-Tetramethyl-6-carboxyrhodamine (TAMARA) on the 3′ end. The TaqMan probe for pirB F1/R1 (5′-TCA CCTGCTGTTGGTTTTCCT-3′) was synthesized and labeled with 6-carboxyfluorescein (FAM) on the 5′ end and TAMARA on the 3′-end. For the assay, TaqMan™ Fast Virus 1-Step Master Mix (Applied Biosystems ™) was used, the final concentration for each primer was 0.2 μM and 0.07 μM for the TaqMan probe at a final volume of 10 μl. The real-time PCR profile was 20 s at 95°C followed by 40 cycles of 1 s at 95°C and 20 s at 59°C. The amplification, detection and the analysis of the data for the real-time PCR assay was carried out with a StepOnePlus PCR system (Applied Biosystems ™).

Three natural known mutant strains of V. parahaemolyticus (pirA and partial pirB deletion), three strains of AHPND causing V. campbelli, one strain of V. shiloi (pirA positive and pirB negative) and ten strains of AHPND causing V. parahaemolyticus were obtained from the Aquaculture Pathology Laboratory bacterial collection. These bacteria were originally isolated from either the gastrointestinal tract of diseased shrimp, water or sediments from AHPND-affected farms in Asia or Latin America during 2013-2018. As negative controls three strains of V. parahaemolyticus without the pirA and pirB genes were used. These Vibrio spp. isolates were used to test the pirA, pirB and internal control primers (18S rRNA and 16S rRNA) for the SYBR Green and the TaqMan real-time PCR assays. All the tested strains were run by triplicate and the mean Ct values and melting temperatures were calculated. The DNA from the bacteria was extracted using the NORGEN Biotek Bacterial Genomic DNA isolation kit following the manufacturer's instructions.

A list of primers that were used for the amplification of pirA and pirB toxin genes. For each primer, the nucleotide sequence, the Tm, the amplicon size and the location on the virulence plasmid in the reference strain V. parahaemolyticus A3 (GenBank accession: KM067908.1) are shown. 

Specific Pathogen Free (SPF) Penaeus vannamei shrimp (average weight 1.0 g) were experimentally challenged via immersion following a previously published protocol [14] . Briefly, V. parahaemolyticus (Strain 13-028A/3) was grown in Tryptic Soy Broth containing 2% NaCl (TSB+) and incubated for 18 h before using for an immersion challenge (CFU 10 6 /ml). The hepatopancreas was dissected out from the moribund animals and recently deceased animals, and it was used for detecting the pathogen via real-time PCR assays. The DNA from the hepatopancreas was extracted using Maxwell-16 ® Cell LEV DNA purification kit (Promega).

To verify there was no cross reactivity with other shrimp pathogens, the multiplex SYBR Green and the duplex TaqMan assays were tested using genomic DNA isolated from P. vannamei known to be infected with white spot syndrome virus (WSSV), Enterocytozoon hepatopenaei (EHP), infectious hypodermal and hematopoietic necrosis virus (IHHNV), necrotizing hepatopancreatic bacteria (NHPB), hepatopancreatic parvovirus (HPV), monodon baculovirus (MBV) and baculovirus penaei (BP).

The DNA fragments for the pirA (80 bp) and pirB (149 bp) were amplified from V. parahaemolyticus 18-408 and cloned into the pDrive Cloning Vector (QIAGEN ® ). The plasmids were designated as VppirA80 and VppirB149. The plasmids were purified using QIAprep ® Spin Miniprep Kit. The sequence of the pirA and pirB fragments was verified by sequencing at the sequencing facility of The University of Arizona, Tucson, AZ.

The sensitivity of the SYBR Green and TaqMan real-time PCR assays were determined using 6-fold serial dilutions of purified VppirA80 and VppirB149 plasmids. The concentrations of plasmid DNA that were utilized ranged from 10 6 to 10 1 copies/μl for all the plasmids. Additionally, ten-fold serial dilutions of 20 ng/μl of total genomic DNA extracted from shrimp hepatopancreas (infected with reference strain A3) were used to determine the detection limit of both the SYBR Green and TaqMan PCR assays. All samples were tested in triplicates.

Three sets of primers listed in Table 1 were tested for compatibility against the internal control primers listed in Table 2 . Only primer set 1 and the internal controls 18S rRNA set 1 and 16S rRNA showed 4 distinguishable melt peaks corresponding to each amplified fragment ( Fig. 2A) . The amplicons for pirA, pirB, 18S rRNA and 16S rRNA from AHPND-infected shrimp 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 (Fig. 2 ).

The real-time PCR results for different strains of bacteria are summarized in Table 3 . The mean Ct and mean melting temperature are shown in Table 4 . When a Vibrio sp. contained both the pirA and pirB genes or one of the two genes, a unique melting curve with unique peak (s) was produced. The pirA negative V. parahaemolyticus strains showed two amplicons, pirB and 16S rRNA, with melting temperatures of 75.20 ± 0.20 and 85.41 ± 0.21°C, respectively (Fig. 2B) . Similarly, the pirB negative strain showed two amplicons, pirA and 16S rRNA, with melting temperatures of 78.21 ± 0.18 and 85.41 ± 0.21°C, respectively (Fig. 2C) . The AHPND causing strains showed three amplicons, pirA, pirB and 16S rRNA, with melting temperatures of 78.21 ± 0.18, 75.20 ± 0.20 and 85.41 ± 0.21°C, respectively (Fig. 2D) .

The duplex TaqMan assay was also able to detect and differentiate when Vibrio spp. contained either pirA or pirB or both pirA and pirB. When Vibrio spp. contained both genes unique amplification curves were detected simultaneously in filter 1 (FAM/pirB) and filter 2 (JOE/ pirA) (Fig. 3) . In contrast, strains that contained only one gene showed a single amplification curve in filter 1 (FAM/pirB) or filter 2 (JOE/pirA) (Fig. 3) . The results for the TaqMan assay for the different strains of bacteria are shown in Table 3 . The SYBR Green assay and the TaqMan assay showed 100% agreement in the results for the detection of the pirA and pirB genes in the axenic Vibrio cultures and the hepatopancreas samples derived from AHPND-infected shrimp (N = 30). Both SYBR Green and TaqMan assays detected pirA and pirB genes in all the samples tested (Table 3 ). In Supplementary Fig. 1-3 . the melt curves for each Vibrio spp. is presented.

The lower limit of detection of the SYBR Green assay and the Table 2 A list of the internal control primers used in the SYBR Green assay for the amplification of Photorhabdus Insect-related (Pir) toxin genes pirA and pirB. For each primer, the nucleotide sequence, the Tm, the product size and the reference is shown. The genes used as internal controls were: β-Actin, Elongation factor1-alpha (EF1-α), Glyceroldehyde-3 phosphate dehydrogenase (GAPDH), 18S rRNA, and 16S rRNA. TaqMan assay for the pirA and pirB amplicons was 10 and 10 copies of recombinant plasmid containing these gene fragments (Fig. 4) . Furthermore, the limit of detection for the pirA and pirB genes in infected shrimp hepatopancreas tissue for both assays was 200 fg of total DNA (Fig. 5) . In Supplementary Fig 4. the melt curves of the serial dilution of DNA from infected tissue is shown. In Supplementary Fig 5. the detection limit (20 pg) of the conventional duplex PCR reported by Han et al. [11] , is shown.

In order to determine the specificity of SYBR Green and TaqMan assays described here, the methods were tested using DNA isolated from P. vannamei shrimp infected with several known viral (WSSV, IHHNV, HPV, MBV, BP), bacterial (NHPB) and fungal (EHP) pathogens. No amplification was obtained in the SYBR Green and TaqMan assays when DNA from other shrimp pathogens were used as a template indicating both assays are specific to Vibrio spp. that contain the pirA and pirB genes.

Acute hepatopancreatic necrosis disease is caused by the Vibrio spp. that harbor a large plasmid with Photorhabdus Insect-Related (Pir) toxin genes pirA and pirB [11] . Both of these genes are necessary for virulence of the bacterium which can be highly lethal to cultured shrimp species of commercial importance. In this study, we developed a multiplex SYBR Green real-time PCR assay and a duplex TaqMan real-time PCR assay for the specific detection of the toxin genes pirA and pirB. The main advantage of both assays is the simultaneous detection and differentiation of the pirA and pirB genes, by utilizing the melt curve analysis for the SYBR Green assay and by using different reporter dyes for the TaqMan assay. To date, these are the only two real-time PCR assays published that can detect and simultaneously differentiate both genes in Vibrio spp.

Recently, Han et al. [3] and Kanrar & Dhar [15] reported the presence of mutant strains of Vibrio spp. that contained either the pirA or pirB gene. In AHPND-causing Vibrio spp., the binary toxin genes are flanked by transposes [11] . It has been proposed that due to a unique genome organization of the V. parahaemolyticus virulence plasmid, it may be lost or acquired by horizontal gene transfer via transposition or homologous recombination [2] . Further evidence of the transfer of these genes between bacteria is reported by Duran-Avelar et al. [10] . These authors detected the first non-Vibrio bacterium, M. luteus, that contains the pirA and pirB genes. It has not been reported if M. luteus is capable of causing AHPND. The discovery of several species of Vibrio and non-Vibrio bacteria carrying the binary toxin genes and causing AHPND suggest that there is an active transmission of the pirA and pirB genes between different bacteria and that these bacteria could acquire or lose pathogenicity. Therefore, the assays reported in this manuscript are fundamental tools for the study of plasmid transmission dynamics and for the detection of mutant strains that contain either pirA or pirB.

Multiplex SYBR Green real-time PCR using melt curve analysis has been previously used for the simultaneous detection of human pathogens (metapheumovirus, rhinovirus, enterovirus and coronavirus), plant pathogens (Plum pox virus), avian pathogens (avian influenza viruses) and bovine pathogens (Clostridium botulinum) [19] [20] [21] [22] [23] . The SYBR Green real-time PCR has been used for the detection and quantification of several shrimp pathogens including IHHNV, WSSV, TSV and YHV [24, 25] . Considering the sensitivity and specificity of the SYBR Green assay when compared with the TaqMan assay and the low cost of the SYBR Green dyes, the method described here is a suitable tool for the detection of Vibrio spp. that cause AHPND and mutants strains that contain either the pirA or pirB gene. Furthermore, the SYBR Green assay described here has two internal controls (the shrimp 18S rRNA and the bacterial 16S rRNA) to avoid false negatives due to the Table 4 Mean Ct and melt temperatures of the different strains of Vibrio spp. and five samples of infected tissue. The poor DNA quality or the inhibition during amplification. Based on the need, the assay can be performed in a single or duplex format for the detection of either one or both toxin genes. Duplex TaqMan real-time PCR has also been extensively used for the simultaneous detection of avian pathogens (avian reovirus and Myoplasma synoviae), human pathogens (Eschrichia coli O157), amphibian pathogens (Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans) and shrimp pathogens (WSSV, Taura Syndrome Virus and PstDNV1) [26] [27] [28] [29] [30] . The sensitivity and specificity of the TaqMan assay was equal to the SYBR Green assay and it can also be used in a single format to detect just one of the two toxin genes. An added advantage of the TaqMan assay is it requires much less time for completion (27 min) since no melt curve analysis is needed making this a valuable tool in detecting Vibrio spp. that causes AHPND as well as mutant strains. However, TaqMan assay is costlier than the SYBR Green assay due to the higher cost of the reagents and the probes.

To summarize, we have developed a multiplex SYBR Green and a duplex TaqMan real-time PCR for the simultaneous detection of the binary toxin genes, pirA and pirB, that are the virulence factors in causing AHPND in shrimp. The current OIE-recommended methods for AHPND detection is based on a one-step conventional PCR [3] and a nested PCR [13] . Therefore, the real-time PCR assays described here represent a valuable tool for detecting AHPND-causing Vibrio spp. The sensitivity and specificity of the assays will reduce time significantly compared to the current OIE-recommended methods.

None.

Roberto Cruz-Flores, Hung Nam Mai, Arun K. Dhar designed the experiments. Roberto Cruz-Flores wrote the manuscript, designed the primers and optimized the conditions of the multiplex SYBR Green realtime PCR and the duplex TaqMan real-time PCR. Roberto Cruz-Flores and Hung Nam Mai prepared the DNA samples. Hung Nam Mai prepared, maintained and extracted DNA from the axenic cultures of the different Vibrio spp. All authors reviewed and approved the final version of the manuscript.

Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp

Correction for Lee et al., the opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin

Characterization and pathogenicity of acute hepatopancreatic necrosis disease natural mutants

Detection of acute hepatopancreatic necrosis disease (AHPND) in Mexico

Report of the FAO/MARD technical workshop on early mortality syndrome (EMS) or acute hepatopancreatic necrosis syndrome (AHPNS) of cultured shrimp (under TCP/VIE/3304), FAO fish

A Vibrio owensii strain as the causative agent of AHPND in cultured shrimp, Litopenaeus vannamei

An isolate of Vibrio campbellii carrying the pir VP gene causes acute hepatopancreatic necrosis disease

Draft genome sequence of non-Vibrio parahaemolyticus acute hepatopancreatic necrosis disease strain KC13.17.5, isolated from diseased shrimp in Vietnam

PirVP genes causing AHPND identified in a new Vibrio species (Vibrio punensis) within the commensal Orientalis clade

Pir A-and pirB -like gene identification in Micrococcus luteus strains in Mexico

Photorhabdus insect-related (Pir) toxin-like genes in a plasmid of Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) of shrimp

Draft genome sequences of four strains of Vibrio parahaemolyticus, three of which cause early mortality syndrome/acute hepatopancreatic necrosis disease in shrimp in China and Thailand

AP4 method for two-tube nested PCR detection of AHPND isolates of Vibrio parahaemolyticus

qPCR assay for detecting and quantifying a virulence plasmid in acute hepatopancreatic necrosis disease (ahpnd) due to pathogenic vibrio parahaemolyticus

Complete genome sequence of a deletion mutant of Vibrio parahaemolyticus from pacific white shrimp ( Penaeus vannamei )

Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data

Validation of reference genes for quantitative measurement of immune gene expression in shrimp

Determination of bacterial load by real-time PCR using a broad range (universal) probe and primer set

Multiplex real-time PCR assay for detection of influenza and human respiratory syncytial viruses

Detection and differentiation of Plum pox virus using real-time multiplex PCR with SYBR Green and melting curve analysis: a rapid method for strain typing

Development of a multiplex real-time PCR assay using SYBR Green 1 chemistry for simultaneous detection and subtyping of H9N2 influenza virus type A

Multiplex real-time PCR SYBR Green for detection and typing of group III Clostridium botulinum

Multiplex SYBR green real-time PCR assay for detection of respiratory viruses

Detection and quantification of infectious hypodermal and hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative PCR and SYBR green chemistry

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

Use of the duplex TaqMan PCR system for detection of shigalike toxin-producing Escherichia coli O157

Development of a real-time multiplex PCR assay for detection of viral pathogens of penaeid shrimp

Duplex real-Time PCR for rapid simultaneous detection of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans in amphibian samples

A duplex real-time PCR assay for the detection and quantification of avian reovirus and Mycoplasma synoviae

Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp

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.

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.mcp.2018.12.004.