The following bacteria strains were used: Mycoplasma (M.) bovis (ATCC 25523), M. arginini (ATCC 23838), M. bovigenitalium (ATCC 19852), M. californicum (ATCC 33461), M. bovirhinis (ATCC 27748), M. alkalescens (ATCC 29103), and M. canadense (ATCC 29418). Additionally, each species of two Mycoplasma strains isolated from cases of naturally occurring mycoplasma mastitis were used for this study. All strains were grown in mycoplasma culture broth (Kanto Kagaku, Japan) at 37℃ for 72 h.

Simplified PCR was performed in a total reaction volume of 20 µL containing 10 µL of 2 × Ampdirect Plus (Shimadzu, Japan), 0.5 U of Nova taq TM Hot Start DNA polymerase (Novagen, UK), 5 pmol of a mycoplasma universal primer set (MycoAce; Nihon Dobutsu Tokusyu Shindan, Japan), and 5 µL of each mycoplasma suspension (10³, 10⁴, and 10⁵ cfu/mL), which were equal to 2.5 × 10², 2.5 × 10³, and 2.5 × 10⁴ cfu/mL. The DNA extraction and purification is unnecessary for this PCR method. PCR was performed in an iCycler PCR System (Biorad, USA). Conditions for the simplified PCR were as follows: initial denaturation at 95℃ for 10 min followed by 35 cycles of denaturation at 94℃ for 30 sec, annealing at 60℃ for 45 sec, and extension at 72℃ for 1 min. The PCR products were separated by electrophoresis on 1.5% (w/v) agarose gels, stained with ethidium bromide, and visualized with a UV transilluminator.

To compare the performance of the simplified PCR with that of standard PCR, we performed a standard PCR assay as previously described [2]. Briefly, DNA from a Mycoplasma suspension was extracted using a DNA extraction kit (Qiagen, Germany) according to the manufacturer's instructions. Standard PCR was performed in a total reaction volume of 20 µL containing 10 × buffer (GE Healthcare, UK), 0.5 U Taq DNA polymerase (GE Healthcare, UK), 4 mM dNTPs, 5 pmol mycoplasma universal primer set, and 5 µL of DNA template which was equal to 2.5 × 10², 2.5 × 10³, and 2.5 × 10⁴ cfu/mL of Mycoplasma. Conditions of the PCR and electrophoresis were the same as those for the simplified PCR described above.

To evaluate the usefulness of the simplified PCR on commercial dairy farms, we compared the sensitivity and specificity of the simplified PCR to that of a culture method. A total of 1,683 quarter milk samples from lactating cows were randomly collected from 18 commercial dairy farms. A total of 159 milk samples were collected from quarters with clinical symptoms such as swelling, induration, and flare. We confirmed that 202 milk samples were collected from quarters that showed no clinical symptoms but had high somatic cell counts (> 400 × 10³ cfu/mL). One hundred µL of milk sample were used to inoculate 2.9 mL of mycoplasma broth (Kanto Kagaku, Japan) and incubated at 37℃ for 72 h. One hundred µL of the broth culture were then plated on a Mycoplasma agar plate (Kanto Kagaku, Japan) and incubated in 5% CO₂ at 37℃ for 14 to 28 days to produce typical Mycoplasma colonies [7]. Each broth culture was analyzed using the simplified and standard PCR as described above.

In this study, we compared the detection rates for seven major Mycoplasma spp. of the simplified and standard PCR assays. All ATCC strains were clearly detected by both methods (Fig. 1). Seven species of two mycoplasma strains isolated from animals with mycoplasma mastitis were also detected by both simplified and standard PCR (data not shown). Gene sequences of PCR-amplified products showed 99% homology with documented sequences in an established gene bank (intergenic spacer region). The minimum detection limit for Mycoplasma by simplified PCR was estimated to be 2.5 × 10³ cfu/mL (Fig. 2). The results obtained from the simplified PCR agreed well with those from standard PCR using purified DNA from broth cultures. Our results showed that simplified PCR for detecting Mycoplasma spp. permits gene amplification without any DNA preparation and performs as well as standard PCR. In this study, we used Amdirect Plus for the simplified PCR to detect Mycoplasma spp. in broth cultures. Ampdirect Plus is a commercially available reagent used for preparing PCR samples without DNA extraction and purification [1]. We have confirmed that the use of Ampdirect Plus reduces labor and time for detecting Mycoplasma spp. in samples.

Out of the 1,685 milk samples cultured in mycoplasma broth, simplified PCR detected Mycoplasma DNA in 152 samples that were also positive according to the culture assay. The concentration of Mycoplasma in these broth cultures was more than 1 × 10⁴ cfu/mL. Four samples were found to be negative by culture and positive by simplified PCR. We speculated that the amplification of DNA from non-viable Mycoplasma in the broth cultures caused the differences between the simplified PCR and culture method results. Two samples were found to be negative by the simplified PCR and positive by culture assay. We confirmed that the concentrations of Mycoplasma in these cultured broths were 2 × 10² and 3 × 10² cfu/mL, which are less than the minimum detection limit of the simplified PCR. Both milk samples were obtained from cows with no clinical symptoms and normal somatic cell counts. Our results suggested that the number of Mycoplasma in the broth cultures of a few milk samples was insufficient for simplified PCR. These samples were not further investigated. The sensitivity and specificity of the simplified PCR method were 98.7% and 99.7%, respectively, of those of the culture assay. Results from the simplified PCR assay completely concurred with those obtained by standard PCR. It has been reported that the sensitivity and specificity of standard PCR for detecting Mycoplasma are 96.2% and 99.1%, respectively, of those of the culture method [2]. In the present study, we succeeded in establishing a simplified PCR method that is able to provide the results identical to those obtained using conventional culture and standard PCR methods.

Standard PCR techniques are labor-intensive and time-consuming, making this method impractical for assaying a large number of milk samples from a commercial dairy farm. This method requires many steps that are quite lengthy; processing about 300 samples requires at least 4~5 h for DNA template preparation and 3 h for completing the PCR cycle. In contrast, 300 samples to be examined within 3 h using the simplified PCR, which does not require several of these laborious steps including Mycoplasma DNA template isolation and purification. Fast and easy screening for mycoplasma mastitis using a simplified PCR assay would enable the quick isolation of infected cows from herds. Our newly developed simplified PCR assay for detecting Mycoplasma spp. is a useful method that can be used to help control and prevent mycoplasma mastitis outbreaks on commercial dairy farms.