Lab Anim Res.  2011 Jun;27(2):141-145. 10.5625/lar.2011.27.2.141.

Sensitive and Specific Detection of Mycoplasma species by Consensus Polymerase Chain Reaction and Dot Blot Hybridization

• Hong S
• Lee HA
• Park SH
• Kim O

• KMID: 2053663
• DOI: http://doi.org/10.5625/lar.2011.27.2.141

Abstract

Mycoplasmas are highly fastidious bacteria, difficult to culture and slow growing. Many species of mycoplasmas are important pathogens that cause respiratory infection in laboratory animals and that are known to affect experimental results obtained with contaminated animals. The aim of the present study was to develop a sensitive and specific assay for the detection of mycoplasma species. To this end, we developed a polymerase chain reaction and dot blot hybridization assay (PCR/DBH) for detecting mycoplasma DNA and evaluated it for its sensitivity and specificity. Mycoplasma consensus primer pairs were used for the amplification of target DNA. When PCR product was visually detected, the limit of detection of the PCR test was 10(2) pg of mycoplasma purified DNA. For DBH, the amplified DNA was labeled by incorporation of digoxigenin (DIG). This DIG-labeled probe was capable of detecting 10(4) pg of purified mycoplasma DNA by DBH. PCR/DBH was more sensitive than PCR or DBH alone and was also very specific. Our PCR/DBH assay can be applied efficiently to confirm the presence of mycoplasma species on clinical samples and to differentiate between mycoplasma species infection and other bacterial infections.

Keyword

Polymerase chain reaction; Dot blot hybridization; PCR/DBH; Mycoplasma

MeSH Terms

Animals
Animals, Laboratory
Bacteria
Bacterial Infections
Chimera
Consensus
Digoxigenin
DNA
Limit of Detection
Mycoplasma
Polymerase Chain Reaction
Sensitivity and Specificity
DNA
Digoxigenin

Figure

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  Figure 1 Gel electrophoresis of amplicons by Mycoplasma hyopneumoniae PCR using consensus primer pairs. Lane 1, Template DNA with 107 pg; 2, Template DNA with 106 pg; 3, Template DNA with 105 pg; 4, Template DNA with 104 pg; 5, Template DNA with 103 pg; 6, Template DNA with 102 pg; 7, Template DNA with 10 pg.

• 

  Figure 2 Hybridization was performed with a digoxigenin-labeled mycoplasma consensus DNA probe. Hybrids were detected by an antibody-conjugate (anti-digoxigenin-alkaline phosphatase conjugate) and by a subsequent enzyme-catalyzed color reaction with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium salt (NBT). 1, Sample DNA with 107 pg; 2, Sample DNA with 106 pg; 3, Sample DNA with 105 pg; 4, Sample DNA with 104 pg; 5, Sample DNA with 103 pg; 6, Sample DNA with 102 pg; 7, Sample DNA with 10 pg.

• 

  Figure 3 PCR/dot blot hybridization. 1, PCR amplicon of 107 pg; 2, PCR amplicon of 106 pg; 3, PCR amplicon of 105 pg; 4, PCR amplicon of 104 pg; 5, PCR amplicon of 103 pg; 6, PCR amplicon of 102 pg; 7, PCR amplicon of 10 pg.

• 

  Figure 4 Specificity of PCR/dot blot hybridization. The mycoplasma consensus probe did not react with other pathogens such as Actinobacillus pleuropneumoniae (AP) and Pasteurella multocida (PM). However, PCR/DBH using a Mycoplasma hyopneumoniae (MP) template DNA resulted in a strong positive signal.

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