A novel polymerase chain reaction assay for the detection of seven Mycoplasma species of cattle origin

The study aimed to develop a pair of polymerase chain reaction primers for detecting ruminant mycoplasma pathogens. We designed a set of primers based on the most similar sequences within 16 S rRNA regions of seven Mycoplasma spp. These primers have high sensitivity for detecting Mycoplasma dispar, M. arginine, M. canadense, M. bovis, M. alkalescens, M. californicum, and M. bovigenitalium within the annealing temperature range of 46 to 48 °C. The minimum amount of DNA that can be detected using the protocol is 250 ng, which is equivalent to 2,000 colony-forming units per mL. The primers can detect mycoplasma from DNA extracted directly from milk samples. The common bovine mastitis pathogens of Staphylococcus aureus coagulase-negative staphylococci, Escherichia coli, Streptococcus uberis, Klebsiella pneumonia, and Kocuria rosea were not detected by the primers. We believe the high sensitivity and specificity of these primers make them useful for detecting infection with seven Mycoplasma species in ruminants, allowing the primers to be used in clinical settings.


Sensitivity test using bulk and mastitis milk samples
Bulk milk samples, either infected with (+) or not infected with (−) Mycoplasma bovis, and milk samples from cows with mastitis, which had been previously identified using primers designed by Bashiruddin et al. 2005, were collected from Taiwan's dairy farms to evaluate the sensitivity of the new set of primers. Milk samples (1 mL) were centrifuged at 3,000 rpm for 10 min, and 100 µL of the liquid supernatant M. bovirhinis, M. alkalescens, and M. canadense (Higuchi et al. 2011); however, because of patent issues, the sequences of the primers have not been published. A loop-mediated isothermal amplification method was developed to detect M. bovis (Higa et al. 2016); however, no method has been developed to successfully detect the majority of ruminant mycoplasma pathogens.
In Taiwan, as no suitable PCR primers have been developed to detect ruminant mycoplasma, there is an urgent need to develop a new set of primers that will enable the investigation of the current prevalence of mycoplasma infection in ruminants. This study aims to develop a set of universal primers for the detection of M. bovis, M. arginini, M. bovigenitalium, M. californicum, M. alkalescens, M. dispar, and M. canadense through PCR.

DNA extraction from standard isolates
This study was firstly conducted in a laboratory in Japan, and completed in a Taiwanese laboratory. The standard isolates from Japan were provided by Dr. Higuchi, Rakuno

Limit of detection
To understand the limit of detection for each standard isolate, a 10-fold series dilution was conducted for the DNA of each standard isolate. Then, 5 µL of each serial DNA dilution was used in PCR. The minimum concentration that could be detected for each amplicon was recorded as the limit of detection.

Specificity tests
To test the primers' specificity, DNA was extracted from Escherichia coli, Streptococcus uberis, Klebsiella spp., Kocuria rosea, Staphylococcus aureus, and coagulase-negative staphylococci, including Staphylococcus epidermidis, S, chromogenes, S. lugdunensis, S. xylosus, and S. simulans. The DNA samples were then used as templates to test the specificity of the primer pair. Based on the sequence of the positive amplicons, this set of primers could not detect any species of Acholeplasma. was collected. DNA from the milk samples was prepared according to the protocol described for the AxyPrep™ Bacterial Genomic DNA Miniprep Kit (Corning Inc.).
Optimization of PCR forMycoplasmaspp. PCR was performed in a total reaction volume of 20 µL containing 2 µL of each forward and reverse primer (10 pmol), 1 µL of double distilled water (DDW), 10 µL of 2X Ampdirect solution (Shimadzu, Japan), and 5 µL of the DNA sample. The PCR conditions used were as follows: initial denaturation at 94 °C for 7 min followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 46 °C for 40 s, extension at 72 °C for 1 min, and then a final extension step of 72 °C for 7 min. The PCR products were separated through electrophoresis on 2% (w/v) agarose gels, stained  Since 2008, an increasing number of outbreaks, which might be related to mycoplasma infections, have been recorded in our laboratory. Therefore, because of the need to establish a PCR assay that detects ruminant mycoplasma pathogens, we developed a useful and practical method for detecting ruminant mycoplasma pathogens. In addition, using these novel primers and the PCR assay, the prevalence of Mycoplasma spp. infection in Taiwan dairy farms could be determined.
The set of primers designed in the current study was based on the sequence of M. bovis, but after analyzing the resulting amplicon, we could differentiate seven Mycoplasma spp. of cattle origin and some Mycoplasma spp. of goat origin (data not published). The primers can detect Mycoplasma spp. in milk directly, without the need for preculturing. Therefore, an outbreak can be detected within half a day. Subsequently, an effective and efficient control and prevention program can be implemented. A recent report described the limits of detection for other PCR assays (Parker et al. 2018). For example, the limit of detection for PCR was 400 colonyforming units per mL (cfu/mL) in broth cultures (Chavez Gonzalez et al. 1995) and 500 cfu/mL in milk samples after DNA extraction (Hotzel et al. 1996). One study reported that infected cattle can shed 10 5 to 10 8 cfu/mL in milk, but 10 3 to 10 6 cfu/mL in milk can be detected in an infected cow before clinical signs become apparent. The same study described that the limit for detection using an ELISA kit was as low as 1,000 cfu/mL in milk after 48 h of incubation . Other assays have been developed to detect Mycoplasma spp. and Acholeplasma spp. contaminants in cell cultures or samples from other animals (Anton et al. 1998). However, many of these assays have disadvantages; for example, one assay requires an enzyme digest system to differentiate the pathogens. Some assays have more complex requirements, such as denaturing gradient gel electrophoresis (DGGE) to differentiate species (McAuliffe et al. 2005). Among the other assays developed so far, we believe that only a few are useful for the detection of mycoplasma pathogens in ruminants. The limit of detection for our set of primers is 250 ng of the DNA sample,

Sensitivity and specificity of the developed primers
An established PCR assay for M. bovis and a newly developed PCR protocol using primers designed in this study were compared. The new primer set was able to detect all M. bovis DNA in all positive samples (Figs. 3,4,5 and 6). The primers did not falsely detect any DNA in the negative samples or any DNA from other bacteria. Therefore, based on our data, the specificity was 100% (Fig. 4).

Discussion
There is a severe lack of research and limited studies on mycoplasma infection in ruminants in Taiwan; M. bovirhinis, M. dispar, and M. alkalescens were first identified in ruminants using biochemical assays in Taiwan in 1982 (data not published); since then, the only research conducted has been a master's degree-level research project that focused on M. capri, and the project was completed in 2020 (Cheng which is equal to 2,000 cfu/mL, and the entire operation can be performed within half a day. Therefore, we believe that our method can be used under any condition and can reasonably compete with other published assays. The limitation of the set of primers must be noted. As this study only tested the main ruminant mastitis pathogens, some opportunistic pathogens, however, may have a chance to bind to the set of primers. Therefore, we would sequence the amplicon every time to avoid misdiagnosis. Although some people may query whether this pair of primers could detect other pathogens, for example, Acholeplasma spp. which has a similar sequence. But when the amplicon has been set, we could identify their differences through their DNA. Using above mentioned Acholeplasma spp. as an example, their similarity was only about 80% (Appendix I). Furthermore, it would be also helpful to culture the pathogen, or to use high resolution melting curve analysis to avoid the mistakes (Al-Farha et al. 2018). In the future, we hope to develop a real-time polymerase chain reaction technique to improve sensitivity and specificity.
In conclusion, the set of primers we designed has high sensitivity to detect seven Mycoplasma species of cattle origin, comprising M. dispar, M. arginine, M. canadense, M. bovis, M. alkalescens, M. californicum, and M. bovigenitalium, within an annealing temperature range of 46 to 48 °C. Some goat mycoplasma pathogens can also be detected. Based on its high specificity and sensitivity, we believe this novel set of primers can help farmers and veterinarians to detect outbreaks in the future.