Development of a novel real-time polymerase chain reaction ( PCR ) assay by amplification of double target genes for quantitative detection of Mycoplasma gallisepticum

Mycoplasma gallisepticum (MG) is the most pathogenic and economically significant pathogen in poultry worldwide. Detection of MG is the first and important step for controlling its transmission. A novel real-time polymerase chain reaction (PCR) assay targeting two conservative genes was developed and evaluated carefully in the study. The 16S rRNA and fMG-2 gene fragments were cloned into PCR 2.1 vectors, and recombinant plasmids (r16S21 and rFmg21) were evaluated by PCR, double digestion and nucleotide sequencing. SYBR green real-time PCR was developed using two purified plasmids as templates, and the amplification conditions and reaction systems of real-time PCR were optimized based on specificity, sensitivity and repeatability. A pair of standard curves were assembled for the realtime PCR by detecting two target genes 16S rRNA and fMG-2. The real-time PCR is highly specific to the target genes, with a detection limit of 9 copies/μl. The result of reproducibility shows that the real-time PCR remained consistent. The result of clinical samples demonstrated that the detection rate of the assay was significantly higher than that of the conventional PCR. The double target genes real-time PCR was highly specific, sensitive, and reproducible and could be used on clinical samples from commercial chickens.


INTRODUCTION
Mycoplasma gallisepticum (MG) is an important infectious respiratory pathogen within chicken farming plant (Leigh et al., 2010).It can trigger chronic respiratory disease (CRD) in chickens and cause serious economic losses for poultry farming (Osman et al., 2009).CRD shows a variety of symptoms including rales of respiratory tract, cough, tracheitis, air sacculitis and maudlin, which often reduce the feed and egg production efficiency (Raviv et al., 2008).MG can be transmitted horizontally by direct or indirect contact manner through the respiratory tract.However, this pathogen does not survive outside of the host for extended periods *Corresponding author.E-mail: Huyuanqing1979@163.com.Tel: +86 596 2528735.Fax: +86 596 2528735.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License  (Sprygin et al., 2011).Mortality due to CRD is lower than other avian diseases, but the infection rate is very high as compared with other diseases.Within intensive livestock farming, infection by CRD often triggers Newcastle disease, avian influenza, infectious bronchitis and colibacillosis, these diseases may lead to more severe problems (Stipkovits et al., 2012).
To date, the accurate and rapid diagnosis for MG is essential to prevent and control the infection by this pathogen.Many detection methods were developed and researched recently, including serology, culture, and molecular identification (Garcia et al., 2005;Ghorashi et al., 2010;Kahya et al. 2010;Zhang et al. 2011).Although isolation of mycoplasmas is the gold standard way for determining MG, time-consuming for this method limits its wide application within poultry production (Levisohn and Kleven, 2000).Serological assays for detecting MG show higher efficient and rapid, such as plate agglutination test, haemagglutination inhibition and enzyme-linked immunesorbent assay (ELISA) (Kempf and Gesbert, 1998;Kempf et al., 1994), but these methods have other limitation for detecting MG infection, including hysteresis of antibody generation and cross-reactions with other pathogens (Kempf et al., 1997).DNA detection is an alternative to conventional culture and serology, of these methods realtime PCR is a more sensitive and rapid assay than conventional PCR detection (Mekkes and Feberwee, 2005).In this study we report the development of a highly specific and sensitive SYBR green real-time PCR assay for detecting 16S rRNA and fMG-2 genes of MG.The novel method was embodied for detecting double conservative genes.

MG strains and DNA preparation
Six mycoplasma strains were used in this study: M. gallisepticum S6 strain was used as template for development of real-time PCR, Mycoplasma synoviae WVu 1853 strain, Mycoplasma iowae 695 strain, Mycoplasma hyorhinis BTS-7 strain, Acholeplasma oculi PG 8 strain, Mycoplasma arginini G230 strain (China Institute of Veterinary Drug Control), Escherichia coli O157:H7 strain, Salmonella enteritidis C50041 strain, and Vibrio parahaemolyticus VP12 strain (our lab) were used to validate the specificity of realtime PCR.The genomic DNA was extracted (DNeasy Blood and Tissue Kit, Qiagen®, of Hilden, Germany) following the manufacturer's instructions from the culture these bacteria, and aliquots of the DNA were stored at -80°C for real-time PCR assay.

Primers for real-time PCR
The real-time PCR assay was designed based on the sequences of 16S rRNA (FJ468422) and fMG-2 (AF075588) genes of MG S6 strain.The primers were designed with primer 3.0 online (http://www.simgene.com/Primer3),and the specificity of primers was assessed using BLAST online (http://www.ncbi.nlm.nih.gov/BLAST/).The cloning primers were designed using primer 5.0 software (Premier Inc., Canada).The primers used are given in Table 1.

Preparation of standard DNA controls
16S rRNA and fMG-2 genes were cloned using specific primers from MG S6 strain.The reaction was carried out in a 25 μl reaction mixture containing 2.5 μl of 10xPCR buffer, 1.0 μl of dNTPs (2.5 pmol•μL -1 ), 0.5 μl of Taq DNA polymerase enzyme (5 U•μL -1 , TaKaRa, Dalian, China) and 3 μl of template, primers to a final concentration of 0.2 μM and nuclease free water was added to make 25 μl.The reaction conditions used are 95°C for 15 min; and 30 cycles of 95°C for 30 s followed by the reaction specific annealing 46°C for 1 min and extension 72°C for 50 s, and finally extension at 72°C for 5 min.The fragments identified as of 16S rRNA and fMG-2 genes were purified using MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0 ® (TaKaRa, Dalian, China).Fragments cloned were ligated into vector pCR 2.1 vector (named r16S21 and rFmg21) and transformed into competent E. coli DH5α cells.Finally, E. coli DH5α cells were cultured on Luria-Bertani plates containing X-gal and IPTG; the white colonies were selected and analyzed using PCR and digested using BamH and Xho for r16S21 plasmid, Xho and Hind  for rFmg21 plasmid.The plasmids of r16S21 and rFmg21 were subjected to DNA sequencing in Genscript® (Genscript Inc.Nanjing, China).

Standard curves for Real time PCR
The standard curves were drawn by real-time PCR, which was performed using templates of 16S rRNA and fMG-2 recombinant plasmids.The reaction system was a 20 μl mix containing 10 μl of SYBR Premix Ex Taq Ⅱ 2xMix (TaKaRa, Dalian, China), 0.4 μl of Rox Reference Dye Ⅱ (TaKaRa, Dalian, China), primers to a final concentration of 0.4 μΜ, 6 μl of water, and 2 μl of template (40 ng/μl).Each real-time RT-PCR reaction was performed using a Gene Amp 7500 thermocycler (Applied Biosystems, Carlsbad, California, USA) with the following PCR conditions: 50°C for 2 min, followed by 40 cycles of denaturation at 95°C for 30 s and annealing at 60°C for 34 s.The quantitation and detection limit of real-time PCRs were determined by 3 independent runs of each reaction, using 10-fold serial dilutions (10 9 to 10 5 copies per reaction) of r16S21 and rFmg21 plasmid as templates.The standard curves were generated by plotting the mean C T values vs. log 10 of the plasmid copy numbers of the three independent runs.Fragments of 16S rRNA and fMG-2 were ligated into vector pCR 2.1 vector, and PCR were performed to evaluate the recombinant plasmids.The result of Figure 1A shows that fragments of 233 and 561 bp were amplified for 16S rRNA and fMG-2, respectively.The Figure 1B and 1C shows the results of restriction enzyme digestion, 16S rRNA by using BamHⅠ and XhoⅠ, fMG-2 by HindⅢ and XhoⅠ.

Specificity test
To verify the specificity of the developed real-time PCR assay, each reaction was performed using genomic DNA extracts from microorganisms including M. gallisepticum S6 strain, M. synoviae WVu 1853 strain, M. iowae 695 strain, M. hyorhinis BTS-7 strain, Acholeplasma oculi PG 8 strain and M. arginini G230 strain, E. coli O157 strain, Salmonella pullorum S06004 strain, Campylobacter jejuni NCTC 11168 strain, V. parahaemolyticus isolate VP12 and mixtures of all these organisms.The real-time PCR were performed according to the procedure and setup as mentioned above.

Sensivity test
The genomic DNA was extracted (DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany) from M. gallisepticum S6 strain, and its concentration was determined using BioPhotometer plus (Eppendorf Inc., Germany).Serial ten-fold dilutions of template were made for confirming the minimum detection limit of the realtime PCR assay developed.Any reaction whose C T value was under 35 was considered positive and higher C T values were considered negative.

Reproducibility test
For the reproducibility of the real-time PCR assay, the test of variation within-run and between-run were performed for 3 reactions using recombinant plasmids (r16S21 and rFmg21) as templates.

Application of the real-time PCR for clinical samples
In this study, 30 samples of chicken swabs were collected from five farms (six swabs per farm) in Anhui province, from birds showing clinical symptoms of chronic respiratory disease (CRD).Real-time PCR were used to detect MG in these samples and normal PCR assay as control.10 samples from apparently healthy birds were used as negative controls.

Standard DNA controls
The 16S rRNA and fMG-2 genes were cloned from M. gallisepticum S6 strain, and these 2 recombinant plasmids were determined by using PCR, double digestion (Figure 1) and DNA sequencing array (data not showed).

Standard curves
Standard curves were made for the real-time PCR by detecting 2 target genes (16S rRNA and fMG-2).Each reaction's standard curve was determined by 3 independent runs of each reaction using 10-fold serial dilutions (10 9 to 10 5 copies per reaction) of the standard DNA control for 2 target genes.The mean C T values, the linear equations and the R-squared values of the realtime PCRs standard curves are shownin Table 2.The curves were depicted as Figure 2, and constructed by 7500 software, version 2.0.1 (Applied Biosystems, Carlsbad, California, USA).

Specificity of the real-time PCR
The results of real-time PCR specificity are given in Figure 3A and 3B.The reactions were positive by detecting genomic DNA of M. gallisepticum S6 strain and mixtures of these 10 organisms; other reactions were negative including those of M. synoviae WVu 1853 strain, M. iowae 695 strain, M. hyorhinis BTS-7 strain, Acholeplasma oculi PG 8 strain, E. coli O157 strain, S.   pullorum S06004 strain, C. jejuni NCTC 11168 strain, V. parahaemolyticus isolate VP12 and M. arginini G230 strain.These results show that the real-time PCR assay is highly specific to the target genes and no crossreactivity was observed for other control microorganisms.

Sensitivity of the real-time PCR
The concentration of genomic DNA of M. gallisepticum S6 strain was 40 μg/μl; amount to 9.25×10 7 copies/μl.
The sensitivity for M. gallisepticum was determined using genomic template DNA extracted from nine 10-fold dilutions of M. gallisepticum S6 strain.The detection limit of the developed real-time PCR was 9 copies/μl (Figure 4A and B).

Reproducibility of the real-time PCR
The reproducibility of the real-time PCR assay was determined through the test of variation within-run and The sensitivity of real-time PCR were determined for 2 target genes, 16S rRNA (Figure 4A) and fMG-2 (Figure 4B), and genomic template DNA were extracted from nine 10-fold (Curves 1 to 9) dilutions of M. gallisepticum S6 strain (9.25×10 7 copies/μl).
between-run using recombinant plasmids (r16S21 and rFmg21) as templates.All reactions remained consistent, which shows that the real-time PCR is repeatable (data not shown).

Detection of the real-time PCR for clinical samples
Out of 30 suspected samples, 17 (56.7%)were positive by real-time PCR, and six samples (20%) were positive through conventional PCR assay.There are no positive out of the 10 samples from chickens without symptoms by using both methods.These results show that the developed real-time PCR has higher sensitivity accuracy and have applicability on clinical samples.

DISCUSSION
In China, MG is the most economically significant poultry mycoplasma, and gives rise to respiratory disease in chickens, turkeys and other avian species.Control of CRD has generally been based on the eradication of the pathogen from breeder flocks and the maintenance of mycoplasma-free status in the breeders and their progeny by biosecurity practices.Serology is the primary method for flock supervisory screening.Sera commonly are examined for antibodies using the serum plate agglutination (SPA) test, a hemagglutination-inhibition (HI) test and an enzyme-linked immunosorbent assay (ELISA) test (Ben Abdelmoumen Mardassi et al., 2008;Raviv et al., 2008).The SPA test is rapid, sensitive and inexpensive but may generate nonspecific results.The HI test is less sensitive but more specific than the SPA test.
The ELISA test is more sensitive than the HI test and more specific than the SPA test.However, many studies showed that serological assays have some drawbacks (Sprygin et al., 2010).The primary goal of the present study was to develop a real-time PCR approach with double target genes to detect M. gallisepticum sensitively in a quantitative manner from clinical setting.Genes 16S rRNA and fMG-2 were two important markers for detecting MG.Sequence data of the 16S rRNA genes have proved to be very useful in studying the phylogeny of MG (Olsen and Woese, 1993).The 16S rRNA nucleotides are convenient for rapid sequence analysis and the presence of universal regions enables its amplification by PCR for subsequent analysis.Another important advantage of the 16S rRNA gene is that it is not prone to horizontal gene transfer (Raviv et al., 2007), and that polymorphism between copies of the gene in the chromosome are not common, due to recombination by gene conversion.Gene of fMG-2 was a conservative marker for detecting MG, some developed PCR approach based on this gene have higher specificity.In this study, these 2 genes were determined separately using real-time PCR.
At present, some MG species-specific PCR, MG realtime PCR, PCR-RFLP and oligonucleotide probe techniques are available (Callison et al., 2006;Jarquin et al., 2009;Kahya et al., 2010;Lierz et al., 2008;Mardassi et al., 2005;Raviv et al., 2008;Sprygin et al., 2010).During the acute stage of the infection the number of MG in the upper respiratory tract is high, but in chronic infection the number of organisms is much lower and routine methods may not detect it (Levisohn and Kleven, 2000).In some situations it may be very difficult to isolate pathogenic mycoplasma consistently from infected flocks.These instances include chronic MG and MS cases and infections with strains of low pathogenicity (Lam and Lin, 1984).The present assay can be applied to the detection M. gallisepticum from clinical samples, and its accuracy was higher than other molecular methods for based on 2 target genes.The specificity and sensitivity of the realtime PCR were evaluated, the results show that MG can be differentiated from other micrograms, including of M. synoviae WVu 1853 strain, M. iowae 695 strain, M. hyorhinis BTS-7 strain, Acholeplasma oculi PG 8 strain, E. coli O157 strain, S. pullorum S06004 strain, C. jejuni NCTC 11168 strain, V. parahaemolyticus isolate VP12 and M. arginini G230 strain.The detection limit of the developed real-time PCR was 9 copies/μl.The real-time PCR assay was evaluated for the ability to detect MG in clinical specimens using 30 samples, and the results indicated that the developed real-time PCR has higher sensitivity than conventional PCR assay on clinical samples.In summary, we have developed a sensitive and specific real-time PCR assay for the detection of the M. gallisepticum in clinical samples from commercial poultry.

Figure 1 .
Figure1.Results of PCR evaluation and restriction enzyme digestion for recombinant 16S rRNA and fMG-2 plasmids.Fragments of 16S rRNA and fMG-2 were ligated into vector pCR 2.1 vector, and PCR were performed to evaluate the recombinant plasmids.The result of Figure1Ashows that fragments of 233 and 561 bp were amplified for 16S rRNA and fMG-2, respectively.The Figure1Band 1C shows the results of restriction enzyme digestion, 16S rRNA by using BamHⅠ and XhoⅠ, fMG-2 by HindⅢ and XhoⅠ.

Table 1 .
Primers used for real-time PCR analysis and gene cloning.

Table 2 .
Summary of the mean C T values, the linear equations and the R-squared values of the real-time PCRs standard curves.