Loop-Mediated Isothermal Amplification for Detection of Staphylococcus aureus in Dairy Cow Suffering from Mastitis

To develop a rapid detection method of Staphylococcus aureus using loop-mediated isothermal amplification (LAMP), four specific primers were designed according to six distinct sequences of the nuc gene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was 1 × 102 CFU/mL and that of PCR was 1 × 104 CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection of Staphylococcus aureus has many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection of Staphylococcus aureus.


Introduction
Cow mastitis is the most common disease in world dairy industry and has made a tremendous economic loss. It not only reduces the milk yield and the quality of milk but also endangers human health and even leads to complete loss of lactation function of dairy cows [1]. Especially the subclinical mastitis is seriously harmful to dairy industry because it has no obvious clinical symptoms and has high incidence. There are more than 20 kinds of pathogens that can cause the cow mastitis, among which the most common one is Staphylococcus aureus [2].
The microbiological assay of milk is considered as "gold standard" of the diagnosis of subclinical mastitis. The conventional method depends on the milk microbial culture and biochemical identification; this method is complicated and time consuming and it has many limitations in practice application. Along with development of molecular biology, the genome sequencings of many bacteria have been completed. In recent years, the PCR detection method based on the sequence of bacteria rRNA regions has been widely applied in clinical diagnosis of medicine and veterinary [3]. However, the PCR method still has some disadvantages such as complex operation, requiring special heating cycle equipment, and so on.
The LAMP technology was developed by Notomi et al. [4]. It is a novel DNA amplification method and can amplify target gene under isothermal conditions with high efficiency, specificity, and sensitivity. Moreover, LAMP only requires simple reaction equipment, such as water bath. At present, LAMP method has been widely applied in detection of viral, bacterial, parasitic, and other pathogens [5][6][7][8]. In this study, we designed a set of specific primers according to the conserved regions of nuc gene of Staphylococcus aureus and developed a highly sensitive and specific LAMP method for detecting clinical samples.  The milk samples were collected from dairy cows suffering from mastitis which had no treatment history. Before collection, the mammary skin of dairy cow was cleaned with warm water, the papilla was disinfected with 75% alcohol, and the collected milk from each papilla was discarded at first three times. The milk samples were collected with 10 mL sterile centrifugal tube and labeled number and date. Then the collected milk samples were frozen with ice bag and taken back to laboratory for use. The nuc gene sequences of Staphylococcus aureus (accession no. EF529606) were selected from the NCBI database. After multiple sequence alignment, four primers were designed using LAMP primer design software (Primer-Explorer, http://www.Netlaboratory.com/) according to the conserved regions of nuc gene. All primers sequences for LAMP are listed in Table 1    green is observed by naked eye under natural light, indicating that the reaction is positive. After reaction, a total of 7.0 µL LAMP products were analyzed by 1.5% agarose gel electrophoresis at 80 V for 50 min. After electrophoresis, the gels were subjected to staining for 10 min by using EB and observed by using a gel imaging system.

Specificity Test.
A total of 40 milk samples were collected from dairy cow suffering from mastitis, and then they were detected by using the developed LAMP. In addition, the DNA of Escherichia coli, Streptococcus agalactiae, Salmonella typhimurium, and Staphylococcus epidermidis were extracted and the specificity test was carried out according to the developed LAMP. The specificity of LAMP was determined by gel electrophoresis according to the specific ladder bands. The result was compared with the specificity of PCR. The samples of LAMP positive reaction were identified by biochemical tests after cultured.

Sensitivity Test.
Taking Staphylococcus aureus as test object, the bacterial liquids were diluted to 1 × 10 6 , 1 × 10 5 , 1 × 10 4 , 1000, 100, and 10 CFU/mL, and then the DNA extraction were carried out. The 10-fold serial diluents of DNA were used as template for LAMP and PCR amplification, respectively. The detection rate and the minimal detectable concentration of LAMP were compared with those of PCR, respectively.

Visualization of LAMP.
A lot of repeat sequences containing target DNA will be amplified when the target DNA is existing in the LAMP reaction system. Thus, the positive control tube and the sample tubes turned green after adding SYBR Green l due to the presence of LAMP products (target DNA), while the negative control tube remained orange (Figures 2(a) and 2(b)). The LAMP assay produced specific ladder bands on the gel (Figures 3 and 4(a)).

Specificity of LAMP. The Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, Salmonella typhimurium,
and Staphylococcus epidermidis were detected by using the developed LAMP, respectively. The results showed that the amplification product was positive only when Staphylococcus aureus was detected. Although the time of isothermal amplification was increased to 1 h, the DNA of other strains could not be detected. These demonstrated that the LAMP method had high specificity for detecting Staphylococcus aureus ( Figure 3).

Sensitivity of LAMP.
The 10-fold serial diluents of DNA extracted from Staphylococcus aureus was used as template for LAMP and PCR amplification, respectively. As shown in Figures 4(a) and 4(b), after gel electrophoresis, the LAMP products produced a ladder band while the PCR products produced one band at 420 bp. The minimal detectable concentration of LAMP was 1 × 10 2 CFU/mL and that of PCR was 1 × 10 4 CFU/mL.

Evaluation of LAMP.
A total of 40 milk samples were collected from dairy cow suffering from mastitis, and the Staphylococcus aureus was detected by using the developed LAMP method and conventional PCR method, respectively. Results showed that the detection rate of LAMP was 100% and that of PCR was 85%; the minimal detectable concentration of LAMP was 1 × 10 2 CFU/mL and that of PCR was 1 × 10 4 CFU/mL. The sensitivity of the LAMP was 100 times higher than that of PCR. At the same time, the results of LAMP reaction were consistent with the results of biochemical tests.

Discussion
Staphylococcus aureus is the main pathogens causing mastitis in dairy cows. It is also one of the main pathogens causing food contamination and thereby leading to food poisoning. At present, the food poisoning caused by Staphylococcus aureus has increasingly become a global public health problem and posed a serious threat to human safety and health [9][10][11]. Currently, there are many methods for detecting Staphylococcus aureus such as the conventional isolation, culture and biochemical identification, immunological detection, and molecular detection. The conventional method is complicated and time consuming and its sensitivity is low. The immunological method has low specificity and sensitivity. The PCR method is sensitive, accurate, and rapid but requires expensive equipment and tedious electrophoresis. Therefore, it does not meet the needs of detection in field and grass-roots sectors and thus limiting the application of PCR method.
The LAMP is a new DNA amplification technology developed by Notomi et al. [4]. This method relies on four specific primers and Bst DNA polymerase that has helicase function, and then the target sequences can be amplified with high efficiency, rapidity, and specificity under isothermal conditions. Four specific primers can recognize the six conserved regions of target gene, ensuring the high specificity and sensitivity of LAMP. The target DNA fragments can be amplified 10 9 to 10 10 times within 1 h under isothermal conditions, indicating that the LAMP is rapid, stable, and efficient [12]. Although there are prone to produce the cross-reaction in LAMP assay, the cross-reaction may be reduced through standardizing operations and some special treatment such as strict experimental partition, adding Dutp and uracil-N-glycosylase (UDG) in reaction system, and disinfecting experimental equipments with alcohol or ultraviolet.
In this study, the LAMP for detecting Staphylococcus aureus is more sensitive than PCR. The positive result can be observed by naked eyes and determined according to the green fluorescence. Therefore, the LAMP is a simple, rapid, specific, sensitive, practical, and visualized detection method of Staphylococcus aureus and it is especially suitable for detection in field and grass-roots sectors.