Development and Application of a Quantitative Competitive PCR Assay for Detecting Mycoplasma hyopneumoniae

Maojun LIU,Dongmei LI,Guodong SU,Yongqi ZHOU,Fangfang BAI,Yuzi WU,Beibei LIU,Guoqing SHAO*

1.Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture/National Center for Engineering Research of Veterinary Bio-products,Nanjing 210014,China;

2.Nanjing Tech-Bank Bio-Industry Co.,Ltd.,Nanjing 211102,China

Responsible editor:Tingting XU Responsible proofreader:Xiaoyan WU

Mycoplasma hyopneumoniae(Mhp)has strict requirements for culture conditions.It is easy to be overshadowed by other mycoplasmas.In the serological detection,serious cross reactions are shown between Mhp and other mycoplasmas,resulting in difficult identification and diagnosis of Mhp[1].PCR technology has become the main method of pathogen detection,and it can be used to detect the microorganisms that cannot be detected by classical isolated culture.In recent years,it has been reported that the Mhp can be detected by nested PCR and fluores cent quantitative PCR[2-3].However,the nested PCR has more complicated procedures (two-step reaction),while the fluorescent quantitative PCR requires expensive equipment in spite of its high accuracy.In this study,based on the 16S rRNA gene of Mhp,one primer pair was designed,and its amplified gene fragment was treated as the target fragment for quantifying Mhp in culture.Since 16S rRNA gene was not Mhp-specific gene,another Mhp-specific primer pair P36 was de signed for identifying Mhp[4].One pair of primers for P36 was used to specifically detect Mhp in the culture.Thus a quantitative competitive PCR assay was established for detecting Mhp in culture,providing real-time monitoring for vaccine production.

Materials and Methods

Materials

Strain and plasmidThe Mhp 168 strain was isolated and preserved by the Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences.The E.coli DH5α was preserved by the Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences.The vector plasmid pMD18-T was purchased from the Takara Biotechnology(Dalian)Co.,Ltd.

Main reagentsThe PCR reagents were purchased from the Promega Corporation.The DNA extraction and purification kit was purchased from the Shanghai Huashun Engineering Machinery Co.,Ltd.The endonuclease Mbo II was purchased from the Takara Biotechnology (Dalian) Co.,Ltd.The lysate A and B was purchased from the Wuxi Institute of Cloning and Genetics Technology.The other reagents were all of analytical grade.

Methods

Preparation of templateThe template was prepared as described by Wu et al[3].

Establishment of P36-based specific detection method for MhpThe sequence of upstream primer P1 was as follows:5’-TTACAGCGGGAAGACC-3’.The sequence of downstream primer P2 was as follows:5’-CGGCGAGAAACTGGATA-3’.The full length of amplified fragment was 427 bp.The PCR reaction system (50 μl) was as follows:10× buffer 5.0 μl,MgCl22.0 μl,dNTPs 3.0 μl,primer P1 0.5 μl,primer P2 0.5 μl,DNA template 2.0 μl,Taq enzyme 0.2 μl,deionized water 36.8 μl.The PCR reaction conditions were as follows:pre-denaturation at 95 ℃for 8 min; denaturation at 80 ℃for 1 min; denaturation at 98 ℃for 1 s,annealing at 49 ℃for 1 min,extension at 72 ℃for 2 min,34 cycles;extension at 72 ℃for 10 min.The amplification products were examined by 1%agarose gel electrophoresis.

Establishment of 16S rRNA-based PCR detection method of MhpThe sequence of upstream primer was as follows:5’-TAGGTCGCAAGCGTTAT-3’.The sequence of downstream primer was as follows:5’-CTCAGGCGGATCATTT-3’.The size of amplified fragment was 343 bp.The PCR reaction system was the same as described above.The reaction conditions were as follows:pre-denaturation at 95 ℃for 8 min; denaturation at 80℃for 1 min;denaturation at 98 ℃for 1 s,annealing at 50 ℃for 1 min,extension at 72 ℃for 2 min,34 cycles; extension at 72 ℃for 10 min.The amplification products were analyzed by 1%agarose gel.

Construction of competitive templateThe sequence of amplified 16S rRNA gene was analyzed with DNAStar.The gene sequences contained two restriction sites for Mbo II.The digested was first digested with Mbo II,and then the fragments were linked with T4 ligase,constructing a gene fragment lacking a sequence of 50 bp.The constructed gene fragment was ligated into pMD18-T vector as an internal control.

Determination of cycle number for exponential growth periodFrom the 20thcycle on,the competitive template was sampled every two cycles till the 40thcycle.Then,the competitive template samples were extended at 72℃for 10 min.The PCR products were examined with 2% agarose gel electrophoresis at the voltage of 120 V.The standard curve of optical density of band to cycle number of template was drawn.Based on the obtained standard curve,the cycle number for exponential growth period was calculated.For the target template,the sampling was conducted every two cycles since the 22thcycle,and it was ended till the 38thcycle.The target template samples were also extended at 72 ℃for 10 min.The following procedures were the same with those of competitive template.

Establishment of quantitative competitive PCR assayThe PCR reaction system(50 μl)was as follows:10×buffer 5.0 μl,MgCl22.0 μl,dNTP 3.0 μl,primer P1 0.5 μl,primer P2 0.5 μl,target template 1.0 μl,competitive template 1.0 μl,Taq enzyme 0.2 μl,deionized water 36.8 μl.The PCR reaction conditions were as follows:pre-denaturation at 95 ℃for 8 min;denaturation at 80 ℃for 1 min; denaturation at 98℃for 1 s,annealing at 49 ℃for 1 min,extension at 72 ℃for 2 min,34 cycles;extension at 72 ℃for 10 min.

Preparation of standard curveIn terms of analysis of competitive amplification results,the optical density value of each lane was analyzed using gel image analyzer and analysis software.The corrected optical density ratio between target band and competitive band of each lane was calculated according to the following formula:

Corrected optical density ratio between target band and competitive band = Optical density of target band/Optical density of competitive band ×Correction value(343/293).

The standard curve of relationship between logarithm of cycle number of competitive template and logarithm of corrected optical density ratio between amplification products of target template and competitive template was drawn.

Repeatability test and stability testEach test was repeated three times so as to verity the repeatability and stability of the quantitative competitive PCR assay.

Drawing of standard curve of relationship between copy number and CCU of MhpThe 1010CCU/ml of Mhp was diluted into 102CCU/ml by 10-fold dilution method.The gradiently diluted internal control plasmid with original concentration of 1.58 ×103μg/ml was treated as the internal control.For each Mhp at a certain CCU/ml,there were two replicates.

Application of quantitative competitive PCR assay in detection of Mhp in cultureThe Mhp was cultured in 10 different vials.The volume of the culture in each of the vials was all 4 ml.After a 10-d culture,the Mhp culture in each of the vials was determined with one vial for one day.On the first day since the starting of the culture,the Mhp culture was orange; the culture turned yellow the next day; in the future days,the culture was pale yellow.The specificity test was carried out using P36-base PCR,and the quantitative detection was carried out using 16S rRNA-based PCR.The gradiently diluted internal control plasmid with original concentration of 1.58×103μg/ml was treated as the internal control.

Results and Analysis

Establishment of P36-based PCR assay

Results of P36-based PCRAs shown in Fig.1,after agarose gel electrophoresis and PCR using P36-based specific primer,a 427-bp band was shown on the gel as expected.

Specificity of P36-based PCRThe established P36-based PCR assay specifically amplified DNA from Mhp but not from any of the other Mycoplasma species or other bacterial species.No false positive amplification was observed,indicating the high specificity of the established assays in electrophoretic analysis(Fig.2).

Sensitivity of P36-based PCRFig.3 showed that the detection limit of the assays was found to be 4.26 ng Mhp genomic DNA.

Establishment of 16S rRNA-based PCR assay

Results of 16S rRNA-based PCRAs shown in Fig.4,a specific 343-bp band was obtained in the products of 16S rRNA-based PCR.

Sensitivity of 16S rRNA-based PCRFig.5 showed that the specific product could be amplified from as low as 0.426ng Mhp genomic DNA using the 16S rRNA primers.

Construction of competitive template

Identification results of competitive templateFig.6 showed that the constructed internal control plasmid had good repeatability.

Sensitivity of PCR for competitive templateAs shown in Fig.7,the sensitivity of established 16S rRNAbased PCR assay reached 3.16 pg.

Determination of cycle number for exponential growth period

Competitive template (internal control)The PCR product of internal control template was increased gradually with the increase of cycle number within 38 cycles(Fig.8).

Target templateThe PCR product of target template was increased gradually with the increase of cycle number within 22-36 cycles(Fig.9).

Establishment of quantitative competitive PCR assay

Optimum Mhp concentration in culture for quantitative competitive PCR assay under optimum conditionsWith a certain amount of target template,with the increased dilution of internal control template by 2 folds,the competitive products of PCR were reduced gradually,but the target product of Mhp was increased gradually.Similarly,with the increased dilution of 10-fold diluted internal control template by 2 folds,the competitive products of PCR were also reduced gradually,but the target product of Mhp was increased gradually(Fig.10).

Preparation of standard curveThe logarithm of concentration of competitive template was treated as the abscissa (X-axis),and the logarithm of corrected optical density ratio between amplification products by competitive template and target template was treated as the ordinate (Y-axis).Thus the regression equation was obtained:Y=-0.682 6X+1.737 3(R=0.97).When Y=0,the corrected optical density ratio between amplification products by competitive template and target template was 1,and at that time,the total DNA amounts of target template (343 bp) is equivalent to the competitive template (293 bp).According to the concentration of competitive template(X value),the original concentration of target template in the quantitative competitive PCR system could be deduced[5].When Y=0,the corresponding X value could be transformed into the copy number of plasmid,which was also the copy number of Mhp.When Y=0,the copy number of competitive template was calculated as follows:101.7373/0.6826,1.526 ×10-9=3.51 ×1011(copies).

Repeatability and stability

The three tests of the established quantitative competitive PCR assay showed high repeatability with variation coefficient of 0.52%,indicating that the established assay was stable and reliable.

Generation of standard curve of relationship between CCU and copy number of Mhp

P36-based PCR results of Mhp with different CCUAs shown in Fig.11,the amplification product of Mhp by P36-based PCR was reduced gradually with the decrease of CCU.The P36-based PCR assay could amplify the Mhp of which the CCU was as low as 105.

Preparation of standard curves of relationship between CCU and copy number of MhpThe Mhp cultures with 1010-102CCU were amplified by 16S rRNA-based PCR.The corresponding copy numbers were calculated.Then the standard curve of relationship between CCU and copy number of Mhp was generated(Fig.12).

Discussion

Color change unit (CCU) is a standard method for quantifying Mycoplasma.However,the CCU assay requires several days,and it has strict requirements for operation.Moreover,the detection results are directly affected by medium,cleanliness of vessels,operation and other human factors.The PCR assays for detecting antigen target genes include bacteriophage technique,limiting dilution method,internal control competition assay and fluorescence quantitative techniques[6-9].The first two methods are generally used for semi-quantification due to their complicated operation and poor accuracy.Although the fluorescence quantitative techniques have high accuracy,it requires high costs and also has high requirements for equipment,so it is unsuitable for clinical testing.The semi-quantitative PCR deduces the content of original template according to the content of product.Among many quantitative PCR assays,only the quantitative competitive PCR assay can obtain two bands simultaneously with only one primer pair under the same conditions.In this study,using the two restriction sites,at a distance of 50 bp,of Mbo II enzyme in the gene of target template,the competitive template lacking a 50-bp fragment compared with that of the target template was constructed,thus the reference caused by the gene itself was reduced in the PCR process.In the PCR reaction,the competitive template and the target template utilized the same reaction system,reducing the difference in non-internal control template among different amplification tubes.

The primer pair used in this study for amplifying target fragment was designed based on the 16S rRNAsequence of Mhp registered in the Gen-Bank.Since the 16S rRNA gene is not specific for Mhp,another primer pair for the specific cytosolic protein P36 in Mhp was designed.The specificity of LDH (P36) was confirmed by PCR method[8].P36 also does not cross-react with other mycoplasmas,LDH in pig,E.coli or Bacillus stearothermophilus.For the quantification of Mhp,the genomic DNA was firstly amplified by P36 primers so as to identify the presence of Mhp.In the quantitative competitive PCR,the gel was stained with EB,and the optical densities of amplified bands of target template and competitive template were compared using the gel imaging system.Then the copy number of tested template was calculated.The established quantitative competitive PCR assay does not require high-value real-time PCR instrument,so it shall provide convenience for clinical laboratory testing.

In this study,a quantitative competitive PCR assay is established for detecting Mycoplasma hyopneumoniae.It provides a simple method for the quantification of clinical samples and laboratory cultures,and also provides a monitoring tool for the Mycoplasma vaccine production.

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