Table 1.
Overview of the characteristics of the PCR methods described for use in diagnosing Mycoplasma species in cattle
| Conventional PCR | Real time PCR SYBR green dye | Real time PCR fluorescent reporter probe | |
|---|---|---|---|
| Overview | Measures the amount of PCR product at the end of the PCR, therefore requires gel electrophoresis to visualize amplified DNA | Measures PCR amplification as the PCR occurs | Measures PCR amplification as the PCR occurs |
| Is semiquantitative through comparison of gel band intensities | Is quantitative | Is quantitative | |
| Dye binds to all double stranded DNA. As the quantity of target double stranded DNA increases, the amount of light emitted by the dye increases proportionally. | Probe binds to a specific targeted region internal to the primer binding sites. As the PCR cycles progress, the reporter dye is cleaved off and fluoresces. An increase in PCR product targeted by the probe causes a proportional increase in fluorescence. | ||
| Increase in background noise because of dye binding to all double stranded DNA | Commercial Assays available | ||
| Specificity | Often targeted 16S rRNA gene, therefore cross‐amplification seen between M. bovis and M. agalactiae 33 | Melt dissociation curve can be used to differentiate between species or nonspecific amplification47 | Alternative target genes and the use of a specific internal fluorescent probe allowed for greater specificity20, 21, 49, 50, 51, 61 |
| Can differentiate amongst Mycoplasma spp. based on gel banding patterns after product digest step,40 or product size on a gel44 | Allows multiplexing to detect multiple targets in a single reaction | ||
| Sensitivity | ∼102‐103 cfu/mL in milk44 | Less commonly used to detect mycoplasma in cattle; sensitivity not reported | ∼101 cfu/mL in milk samples for M. bovis, M. californicum, and M. bovigenitalium 49 |
| ∼102 cfu/mL in lung samples for M. bovis 50 | |||
| ∼105, 104, and 107 cfu/mL in semen samples, and ∼106, 104, and 107 cfu/mL in swab samples for M. bovis, M. californicum, and M. bovigenitalium, respectively61 |
Abbreviation: PCR, polymerase chain reaction.