Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=cavp20 Avian Pathology ISSN: 0307-9457 (Print) 1465-3338 (Online) Journal homepage: https://www.tandfonline.com/loi/cavp20 Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum Marcela da Silva Rubio, Rafael Antonio Casarin Penha Filho, Adriana Maria de Almeida & Angelo Berchieri Junior To cite this article: Marcela da Silva Rubio, Rafael Antonio Casarin Penha Filho, Adriana Maria de Almeida & Angelo Berchieri Junior (2017) Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum, Avian Pathology, 46:6, 644-651, DOI: 10.1080/03079457.2017.1339866 To link to this article: https://doi.org/10.1080/03079457.2017.1339866 View supplementary material Accepted author version posted online: 07 Jun 2017. Published online: 17 Jul 2017. Submit your article to this journal Article views: 352 View Crossmark data Citing articles: 2 View citing articles https://www.tandfonline.com/action/journalInformation?journalCode=cavp20 https://www.tandfonline.com/loi/cavp20 https://www.tandfonline.com/action/showCitFormats?doi=10.1080/03079457.2017.1339866 https://doi.org/10.1080/03079457.2017.1339866 https://www.tandfonline.com/doi/suppl/10.1080/03079457.2017.1339866 https://www.tandfonline.com/doi/suppl/10.1080/03079457.2017.1339866 https://www.tandfonline.com/action/authorSubmission?journalCode=cavp20&show=instructions https://www.tandfonline.com/action/authorSubmission?journalCode=cavp20&show=instructions http://crossmark.crossref.org/dialog/?doi=10.1080/03079457.2017.1339866&domain=pdf&date_stamp=2017-06-07 http://crossmark.crossref.org/dialog/?doi=10.1080/03079457.2017.1339866&domain=pdf&date_stamp=2017-06-07 https://www.tandfonline.com/doi/citedby/10.1080/03079457.2017.1339866#tabModule https://www.tandfonline.com/doi/citedby/10.1080/03079457.2017.1339866#tabModule ORIGINAL ARTICLE Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum Marcela da Silva Rubio, Rafael Antonio Casarin Penha Filho, Adriana Maria de Almeida and Angelo Berchieri Junior School of Agricultural and Veterinary Sciences, São Paulo State University (FCAV/UNESP), São Paulo, Brazil ABSTRACT Currently there are 2659 Salmonella serovars. The host-specific biovars Salmonella Pullorum and Salmonella Gallinarum cause systemic infections in food-producing and wild birds. Fast diagnosis is crucial to control the dissemination in avian environments. The present work describes the development of a multiplex qPCR in real time using a low-cost DNA dye (SYBr Green) to identify and quantify these biovars. Primers were chosen based on genomic regions of difference (RoD) and optimized to control dimers. Primers pSGP detect both host- specific biovars but not other serovars and pSG and pSP differentiate biovars. Three amplicons showed different melting temperatures (Tm), allowing differentiation. The pSGP amplicon (97 bp) showed Tm of 78°C for both biovars. The pSG amplicon (273 bp) showed a Tm of 86.2°C for S. Gallinarum and pSP amplicon (260 bp) dissociated at 84.8°C for S. Pullorum identification. The multiplex qPCR in real time showed high sensitivity and was capable of quantifying 108–101 CFU of these biovars. ARTICLE HISTORY Received 31 January 2017 Accepted 1 June 2017 KEYWORDS Fowl typhoid; pullorum disease; SYBr green; melting curve; ROD; identification; biovar Introduction Salmonella enterica belongs to the family Enterobacter- iaceae and, currently, 2659 different serovars have been identified (Issenhuth-Jeanjean et al., 2014). All serovars are pathogenic. However, diseases in birds are classified in three different categories. The flagellated serovars, which are the vast majority (e.g. S. Enteritidis and S. Typhimurium), cause paratyphoid infections in differ- ent hosts including humans, mammals, reptiles and birds, with pronounced gastrointestinal colonization (Barletta et al., 2013). Poultry are susceptible to paraty- phoid infections and are frequently associated as source of infection in foodborne disease in humans through the consumption of contaminated meat or eggs (Omiccioli et al., 2009). The other two categories of diseases in birds are fowl typhoid caused by Salmonella enterica serovar Galli- narum biovar Gallinarum (S. Gallinarum) and pull- orum disease caused by Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum). Both bio- vars are closely related genetically, non-motile and are host-specific pathogens of birds (Löfström et al., 2010; Feng et al., 2013). Infections or outbreaks are of compulsory notification to the World Organisation for Animal Health (OIE), due to the mortality of ani- mals, losses caused and risk of dissemination to other countries. S. Pullorum affects newly hatched young chicks, causing septicaemic colonization, with high mortality in affected flocks. Infection initiates mainly from verti- cal transmission originating from subclinically infected adult breeders. However, horizontal transmission con- tributes to fast dissemination within the flock. S. Galli- narum is horizontally transmitted and evidence points to a vertical dissemination route (Celis-Estupinan et al., 2017). Moreover, the infection causes high morbidity and up to 80% of mortality in young or adult chickens (Shivaprasad, 2000). Different factors, such as the interaction with the intestinal epithelium, high inva- siveness, intracellular survival in infected macrophages and evasion of the immune response, are involved in the pathogenesis of these bacteria (Soria et al., 2013). The gold standard method used for the diagnosis of S. Gallinarum and S. Pullorum during outbreaks is microbiological culture, for isolation, biochemical identification and serotyping of the isolate (Santana et al., 2011; Ma et al., 2014). In addition to direct methods used to isolate these bacteria, indirect methods are important for epidemiological studies and monitoring of the sanitary conditions in flocks. Serological tests such as ELISA and rapid sero-aggluti- nation test can detect antibodies against Salmonella spp. and PCR is capable of detecting and identifying the DNA of the pathogen (Andrade et al., 2010). The bacterial culture of Salmonella spp. has a few disadvantages compared to molecular biology tech- niques. Among these, the direct identification of the bacteria is difficult due to the growth of other microor- ganisms present in the sample, especially from © 2017 Houghton Trust Ltd CONTACT Rafael Antonio Casarin Penha Filho rafaelpenha12@yahoo.com.br Supplemental data for this article can be accessed at https://doi.org/10.1080/03079457.2017.1339866 AVIAN PATHOLOGY, 2017 VOL. 46, NO. 6, 644–651 https://doi.org/10.1080/03079457.2017.1339866 http://crossmark.crossref.org/dialog/?doi=10.1080/03079457.2017.1339866&domain=pdf mailto:rafaelpenha12@yahoo.com.br https://doi.org/10.1080/03079457.2017.1339866 http://www.tandfonline.com microbiota. The complete identification depends on biochemical and serological analysis of each isolate, increasing the time and cost to obtain results with this method (Chen et al., 2010). The diagnosis by ELISA technique produces results within two days. However, this technique relies on the detection of anti- bodies and may require confirmation by conventional microbiology, which prolongs obtaining a diagnosis (Dickel et al., 2005). The PCR technique has been successfully estab- lished for fast and reliable diagnosis of different patho- gens, including fastidious bacteria. The results may be obtained within two days or less. However, positive results do not require the confirmation by conventional microbiology. The result analysis follows an established pattern of interpretation (Lin et al., 2011). Many epide- miological surveys were successfully conducted during the last decades using PCR, because this method can be designed for high specificity and sensitivity (Chen et al., 2010). Real-time PCR has been more recently developed for diagnosis of pathogens, with the features of higher sensitivity than conventional PCR and faster protocols to obtain the results. Thus, in the present study, we describe the development of a multiplex real-time PCR to specifically detect and differentiate closely related biovars S.Gallinarum and S. Pullorum and sim- ultaneously quantify the bacterial numbers in samples. Material and methods Bacterial strains for validation of the real-time PCR assay Twenty-eight isolates from biovar S. Gallinarum and 18 isolates from biovar S. Pullorum were used for DNA extraction, development and validation of the multiplex quantitative PCR in real time, including the sequenced strains S. Gallinarum SG9 (accession num- ber: CM001153.1), S. Gallinarum 287/91 (a.n.: AM933173.1), S. Pullorum FCAV198 (a.n.: AZRG00000000). Additionally, DNA from 59 different Salmonella isolates (including non-typhoidal and typhoidal serovars) and six other Enterobacteriaceae (Supplemental Table 1) were used as negative controls for validation of the primers’ specificity. Isolates were obtained from two National Reference Laboratories (Supplemental Table 1) or from the Laboratory of Avian Pathology at the School of Agricultural and Veterinary Sciences (FCAV-Unesp/Jaboticabal/SP) and stored at −80°C. Bacterial DNA extraction Each isolate was cultured in Luria-Bertani broth (LB) at 37°C, shaking at 100 rpm for a period of 18 hours, including the bacteria used as negative controls. Before DNA extraction, all S. Gallinarum and S. Pullorum cul- tures were quantified by plating serial dilutions in Bril- liant Green agar (Oxoid CM0263; Oxoid Ltd, Basingstoke, UK) of the culture with subsequent incu- bation at 37°C for 24 h. After incubation, the bacterial numbers of each culture were evaluated and the colony forming units per ml (CFU/ml) were transformed to Log10. DNA extraction of all bacterial cultures was per- formed using the QIAamp DNAMini kit (Qiagen, Hil- den, Germany) following the manufacturer’s instructions. The purity and concentration of bacterial DNA was analysed by spectrophotometry (Nanodrop 1000 Thermo Fisher Scientific, Waltham, MA, USA). Primer design for differential diagnosis between S. Gallinarum and S. Pullorum Three pairs of primers were used and the sequences and details are in Table 1. The primer pSGP was designed to detect the S. enterica serovar Gallinarum, including both biovars, without detection of other typhoidal or non-typhoidal Salmonella sp. serovars. The primers pSG and pSP were designed to differen- tiate S. Gallinarum biovar Gallinarum (pSG) and S. Gallinarum biovar Pullorum (pSP) based on the analysis of regions of difference (RoDs) from each bio- var (Batista et al., 2015). Primers were designed using the Primer 3 software (Free Software Foundation, Bos- ton, MA, USA), based on the complete genome sequence of S. Gallinarum and S. Pullorum available at the NCBI database (http://www.ncbi.nlm.nih.gov/ genome/) and validated in silico using the primer BLAST tool, to check for non-specific annealing with other sequenced microorganisms. Cloning of the target DNA amplicon The target genome regions used for the specific ampli- fication and diagnosis of each biovar were cloned into plasmids for use as positive controls and to obtain the quantitative standard curve in the real-time PCR. Briefly, the amplicons obtained after conventional PCR using the primers pSG for S. Gallinarum and pSP for S. Pullorum were digested with BamHI and subsequently cloned into plasmids. After cloning, the accurate copy number of the target amplicons was cal- culated and the DNA of each clone was diluted, corre- sponding to 108, 107, 106, 105, 104, 103, 102 and 101 CFU of S. Gallinarum or S. Pullorum, to obtain the quantitative standard curve. Conditions of the qPCR in real time The multiplex real-time PCR was performed using the following optimized reaction mix of 13 µl Lumi- noct® SYBr® Green qPCR Readymix (Sigma-Aldrich, St. Louis, MI, USA), containing 1.25 U of Taq DNA AVIAN PATHOLOGY 645 http://www.ncbi.nlm.nih.gov/genome/ http://www.ncbi.nlm.nih.gov/genome/ polymerase and adding 0.1 µM of the primer pSGP, 0.05 µM of primers pSG and pSP, 2.5 µl of serially diluted DNA or eluted DNA after extraction from tested samples, and ultrapure water to make the final volume 25 µl. The first dilution (10−1) corresponded to 100 ng of DNA and the following dilutions were made in log10 up to 10−8 from the initial DNA sample. The qPCR in real time was performed in the C1000 TouchTM Thermal Cycler (Bio-Rad, Hercules, CA, USA), with cycling protocol starting with one dena- turation cycle at 94°C for 2 min, followed by 40 cycles at 94°C for 15 s and 63°C for 30 s. After amplification, the melting curve was performed to obtain the specific melting temperature (Tm) of each amplicon for the differential diagnosis between S. Gallinarum and S. Pullorum. Standard curve for bacterial quantification Serially diluted plasmid DNA containing the cloned DNA from biovars S. Gallinarum and S. Pullorum was used to prepare the quantitative standard curve. The reactions followed the optimized PCR conditions for the multiplex reactions. The Software CFX Man- agerTM 3.1 (Bio-Rad) was used to calculate the regression coefficients, standard deviation and stan- dard curves efficiency ratio. The linear standard curve was used for quantification of number of genes copies that corresponded to the number of CFU of each biovar. The reactions were acceptable for analysis when R2 value was higher than 0.99. Results and discussion The genus Salmonella spp. has often been reported in studies using different microbiological and molecular techniques for the identification and differentiation in samples from company and food-producing animals (Dickel et al., 2005; Malorny et al., 2007; O’Regan et al., 2008; Mcguinness et al., 2009; Omiccioli et al., 2009; Andrade et al., 2010; Löfström et al., 2010; Park et al., 2011; Barletta et al., 2013; Dobhal et al., 2014; Li et al., 2014; Ma et al., 2014). The fast diagnosis and identification of these bacteria is crucial for the control of bacterial dissemination, reduction of losses in animal production and risks to human health caused by foodborne infections (Chen et al., 2010; Cheraghchi et al., 2014). Currently there are 2659 pathogenic serovars ident- ified and most cause gastrointestinal infections in a large number of hosts (Issenhuth-Jeanjean et al., 2014). However, a restricted number of serovars are host-specific. Considering the high pathogenicity of the host-specific biovars S. Gallinarum and S. Pullorum to birds, the differentiation from others is important to take the correct measures in commercial poultry farms. In Brazil, the presence of either of these two biovars in breeder flocks of chickens condemns the entire flock to elimination, sanitation and replacement of the birds. Thus, the diagnosis has to be fast and accurate, to avoid false positives, as the presence of other non- typhoidal serovars allow treatments and milder biosaf- ety measures in these flocks. Different PCR methods have been described for the differential diagnosis of a few Salmonella serovars, including S. Gallinarum and S. Pullorum and research on this topic is continuously developing (Kisiela et al., 2005; Jeon et al., 2007; Batista et al., 2016; Ren et al., 2017; Xiong et al., 2017). Currently, modern diagnostic tools, such as real-time PCR, have become accessible to researchers and diagnostic laboratories. However, the design of specific probes and primers for this technique is a limitation as it depends on availability of genome sequences and bioinformatics analysis. The present work shows an extensive analysis and validation of a real-time PCR, using all its data, including the melting curve for differentiation of each PCR product by the respective Tm, and consequently for specific diagnosis of each biovar. As shown in Table 1, the sequences of the three primers and the length of each amplicon obtained have different Tm when the melting curve is evaluated, allowing differentiation of the serovar Sal- monella enterica serovar Gallinarum from 2659 other serovars (Issenhuth-Jeanjean et al., 2014), and also the differentiation between the biovars S. Gallinarum and S. Pullorum, which share large homologous gen- ome regions (Batista et al., 2015). The applicability of this reaction by different labora- tories worldwide depends on the costs of the reaction. Thus, we considered all the available DNA fluorescent dyes available and opted to use SYBr Green instead of probes. The use of the SYBr Green system in multiplex PCR in real time has been reported in different studies to identify pathogens, allergens, microbiological viola- tions in contaminated food and antibiotic-resistant genes (Fukushima et al., 2003; Ponchel et al., 2003; Table 1. Specifications of the primers used in the study. Primer Sequence Product size (bp) Concentration (µM) Amplicon Tm pSGP F pSGP R TGGCCTTGACGTTATTCGTT AGCATTTTGGTCACCTGCAA 97 0.1 78.0°C pSG F pSG R ATGGCGAGTCCGCCCAGAGT GGCGGTATGCTGGTTGCCGT 273 0.05 86.2°C pSP F pSP F CCGCCTGCGCGATGGCTTTA TCTGGTTGACGGCGTGGGGA 260 0.05 84.8°C Note: Primers pSG were designed based on ROD43 and primers pSP were designed based on ROD24 (Batista, 2013). 646 M. D. S. RUBIO ET AL. Varga & James, 2005; Liu et al., 2006; Fan et al., 2007; Pafundo et al., 2010; Rajtak et al., 2011; Kagkli et al., 2012;Cheng et al., 2013;Gomes et al., 2014; Singh&Mus- tapha, 2014). However, this fluorescent dye, differently from probe systems, binds indistinctly to double- strandedDNA, emitting interfering levels of fluorescence in case of nonspecific amplification. Despite the in silico validationof theprimers showing a specific site of anneal- ing, themultiplex qPCR in real time described hereinwas developed after extensive titration of the primer concen- trations and validation of annealing temperatures, to avoid nonspecific amplifications and primer dimers. All three primer pairs used in the study were tested and validated with control strains, to determine the optimal concentration for each primer and the appro- priate annealing temperature, in order to reach ampli- fication and prevent the occurrence of non-specific reactions. Based on the results obtained with individu- ally tested primers the multiplex qPCR in real time was performed. The optimized annealing temperature for this reaction was set at 63°C, which offered good con- ditions for the annealing of primers and amplification of the DNA, without the occurrence of any nonspecific fluorescence. The results obtained from the standardiz- ation of individual primer pairs or multiplex qPCR in real time were checked by agarose gel electrophoresis to confirm correct amplification based on the length of the products and the absence of nonspecific reac- tions as shown in Figures 1 and 2, respectively. The results of the multiplex qPCR in real time with positive control DNA samples are shown in Figure 3. For diagnosis of each biovar, one amplification curve is obtained by the multiplex qPCR in real time. However, the differentiation is further performed by the analysis of the melting curve, obtained after ampli- fication. This post-analysis shows the dissociation Tm of each amplicon, which is represented by a peak of flu- orescence loss, when approximately 50% of double- stranded DNA (dsDNA) from amplicons is dissociated at the corresponding temperature and the SYBr Green reaction is lost. The evaluation of the Tm has shown consistency and sensitivity for the purpose of differen- tial diagnosis of bacteria and virus with low mutation rates. Two factors influence the Tm of each amplicon: the length of the dsDNA fragment and the CG content. The higher values of these components require higher Tm for dissociation. The differential diagnosis between biovars in the qPCR in real time was performed using the Tm values. As shown in Figure 3(A) and 3(B) it is possible to notice two different dissociation peaks, cor- responding to two different Tm in the melting curve after the multiplex PCR in real time. The first dis- sociation peak, with Tm of 78°C, refers to the amplicon with 97 bp obtained with primer pSGP, present exclu- sively in biovars S. Gallinarum and S. Pullorum (sero- var S. Gallinarum), but not detected in any other Salmonella serovar. In the same multiplex qPCR also occurred the amplification for differentiation between biovars. As demonstrated in Figure 3(A), biovar S. Gal- linarum is specifically detected by the primer pSG and the corresponding dissociation peak occurs at the Tm of 86.2°C, while the amplicon obtained with primers pSP designed for biovar S. Pullorum has a dissociation Tm of 84.8°C (Figure 3(B)). As carried out with the individual reactions, the efficacy, specificity and quality of the multiplex qPCR in real time were evaluated and confirmed by agarose gel electrophoresis, and the results are shown in Figure 2. The amplicons with 273 bp corresponded to biovar S. Gallinarum and with 260 bp corresponded to S. Pullorum. Figure 1. Agarose gel electrophoresis of the real-time qPCR amplicons using primer pairs separately. (M) Molecular Marker (100 bp); (B) Negative control (blank); (1) Primer pSGP with DNA of S. Gallinarum; (2) Primer pSGP with DNA of S. Pullorum; (3) Primer pSG with DNA of S. Gallinarum; (4) Primer pSG with DNA of S. Pullorum; (5) Primer pSP with DNA of S. Gallinarum; (6) Primer pSP with DNA of S. Pullorum. Figure 2. Agarose gel electrophoresis with amplicons from the multiplex qPCR in real time. (M) Molecular Marker (100 bp); (B) Negative control (blank); (1) Primers pSGP and pSG with S. Gal- linarum DNA sample; (2) Primers pSGP and pSG with S. Pull- orum DNA sample; (3) Primers pSGP and pSP with S. Gallinarum DNA sample; (4) Primers pSGP and pSP with S. Pull- orum DNA sample; (5) Primers pSG and pSP with S. Gallinarum DNA sample; (6) Primers pSG and pSP with S. Pullorum DNA sample; (7) Primers pSGP, pSG and pSP with S. Gallinarum DNA sample; (8) Primers pSGP, pSG and pSP with S. Pullorum DNA sample. AVIAN PATHOLOGY 647 As shown in Figure 2, the duplex qPCR in real time can identify the pathogen at the level of the serovar and the biovar. However, differential diagnosis was achieved by the addition of the third primer pair com- posing the multiplex reaction, as per results shown in lanes 7 and 8. The differential diagnosis of these two typhoidal biovars has been previously reported by con- ventional PCR and methods (Ribeiro et al., 2009; Batista et al., 2013). However, the described techniques require two separate reactions and agarose gel electro- phoresis for biovar differentiation, which affects time and costs for this reaction. In a single reaction the mul- tiplex reaction was able to perform the identification and differentiation between the biovars, based on the specific Tm of the amplicons. The specificity of the multiplex qPCR in real time for differential diagnosis between biovars was tested with DNA samples from non-typhoidal Salmonella. In such cases it is possible to notice the amplicons obtained with primers pSG (273 bp) or pSP (260 bp). However, the amplicon with 97 bp and Tm of 78°C is not present in non-typhoidal Salmonella. Thus, for differential diagnosis between these two typhoidal Sal- monella biovars, it is necessary to include the primer pSGP for the differentiation from other non-typhoidal serovars. Moreover, as shown in Figure 3(C), amplifi- cation only with primers pSG and pSP may be used for diagnosis of other serovar from genus Salmonella, such as S. Enteritidis in cases when the pSGP amplicon with 97 bp (Tm peak of 78°C) is absent. In addition to the amplification with positive diag- nosis obtained with conventional PCR, another advan- tage of diagnosing with PCR in real time is the capacity of quantification of the subject of study. Based on the amplification data plotted, the cycle threshold (Cq) values are informative of the genetic material quantity, corresponding to the bacterial numbers in the sample. In the present study the multiplex qPCR in real time was developed to determine the sensitivity limit for quantification of Salmonella. The standard curve with genomic and plasmid DNA containing the genes of interest for each biovar was prepared. The conven- tional quantification of Salmonella by microbiological culture has a detection limit of 102 CFU/ml and lower bacterial numbers may not be detected without sample enrichment (Malorny et al., 2008). As shown in Figure 4, the standard curve was prepared with seri- ally diluted DNA, and each dilution showed an interval of approximately four cycles for S. Gallinarum and three cycles for S. Pullorum (Table 2), corresponding to the dilution sequence of each biovar. Moreover, it was possible to detect and quantify all DNA samples, including the lowest dilution, corresponding to 101 CFU/ml from both biovars, demonstrating the high sensitivity obtained with the described reaction. Considering the high sensitivity, this diagnostic tool is capable of detecting low numbers of this pathogen, even without enrichment of the sample, consequently reducing time to obtain results. Poultry production is often affected by acute and subclinical infections caused by enterobacteriaceae of genus Salmonella spp. and these bacteria are respon- sible for health impacts and large economic losses worldwide (Dobhal et al., 2014). The outcome of the disease depends on different factors, such as age of birds, genetic resistance and immunosuppressive Figure 3. Melting curves and Tm peaks of the multiplex qPCR in real time for differential diagnosis between S. Gallinarum and S. Pullorum. (A) Tm peaks refer to the amplification of S. Gallinarum DNA samples with primers pSGP and pSG; (B) Tm peaks refer to the amplification of S. Pullorum DNA samples with primers pSGP and pSP; (C) Tm peaks refer to negative results using S. Enteritidis DNA samples in which no amplification with pSGP is noted (Tm 78°C), and amplifications with pSG, pSP occur together. 648 M. D. S. RUBIO ET AL. conditions. The option for treatment or elimination of the flock depends on the serovar. Thus, correct and fast diagnosis is very important for the right decision. Cur- rently, the available techniques used for diagnosis have limitations, considering the time for results and low sensitivity to reduced bacterial loads in organ or environmental samples. Therefore, the proposed mul- tiplex qPCR in real time has shown the capacity to diagnose the two avian host-specific Salmonella biovars in a single reaction. Additionally, a large number of non-typhoidal serovars can be detected in the reaction, differentiating from S. Gallinarum and S. Pullorum. Furthermore, the described technique is also capable of quantifying unknown bacterial loads, in addition to the positive or negative results obtained with con- ventional methods. Overall, the present study demonstrated the possi- bility of using a low-cost DNA dye such as SYBr Green in a convenient multiplex qPCR in real time for identification and quantification of two major typhoidal Salmonella biovars, capable of infecting birds and causing systemic infection and mortality. Thus, the described technique may facilitate the diag- nosis and accelerate protocols to control the spread of these pathogens, which are of worldwide occurrence and which are able to disseminate both vertically and horizontally among avian hosts. Disclosure statement No potential conflict of interest was reported by the authors. Funding The authors thank the Sao Paulo Research Foundation (Fun- dação de Amparo à Pesquisa do Estado de São Paulo – FAPESP) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the financial support. M.S.R. was funded by doctorate scholarship [process number 2014/05496-7] and R.A.C.P.F. was funded by post-doctoral scholarship [process number 2012/24017-7]. References Andrade, R.B., Gemelli, T., Onder, L.P.D., Cristina, K., Brito, T., Barboza, A.A.L. & Brito, B.G. (2010). Métodos diagnósticos para os patógenos alimentares: Campylobacter sp., Salmonella spp. e Listeria monocyto- genes. Arquivos do Instituto Biológico, 77, 741–750. Figure 4. Amplification curves of serially diluted DNA using SYBr Green I (left) and quantitative standard curve (right) obtained by multiplex qPCR in real time for differential diagnosis and absolute quantification of S. Gallinarum and S. Pullorum. (A) S. Gallinarum DNA sample; (B) S. Pullorum DNA sample. Table 2. 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Journal of Virological Methods, 123, 213–220. Xiong, D., Song, L., Tao, J., Zheng, H., Zhou, Z., Geng, S., Pan, Z. & Jiao, X. (2017). An efficient multiplex PCR-based assay as a novel tool for accurate inter-serovar discrimi- nation of Salmonella Enteritidis, S. Pullorum/Gallinarum and S. Dublin. Frontiers in Microbiology 8, 420. AVIAN PATHOLOGY 651 Abstract Introduction Material and methods Bacterial strains for validation of the real-time PCR assay Bacterial DNA extraction Primer design for differential diagnosis between S. Gallinarum and S. Pullorum Cloning of the target DNA amplicon Conditions of the qPCR in real time Standard curve for bacterial quantification Results and discussion Disclosure statement References