Quantitative Polymerase Chain Reaction Coupled With Sodium Dodecyl Sulfate and Propidium Monoazide for Detection of Viable Streptococcus agalactiae in Milk
Streptococcus agalactiae is an important pathogen causing bovine mastitis. The aim of this study was to develop a simple and specific method for direct detection of S. agalactiae from milk products. Propidium monoazide (PMA) and sodium dodecyl sulfate (SDS) were utilized to eliminate the interferenc...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2019-03-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fmicb.2019.00661/full |
| _version_ | 1818575061735440384 |
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| author | Yankun Zhao Yankun Zhao Yankun Zhao He Chen He Chen He Chen Huimin Liu Huimin Liu Huimin Liu Huimin Liu Jianxing Cai Jianxing Cai Jianxing Cai Lu Meng Lei Dong Nan Zheng Jiaqi Wang Cheng Wang Cheng Wang |
| author_facet | Yankun Zhao Yankun Zhao Yankun Zhao He Chen He Chen He Chen Huimin Liu Huimin Liu Huimin Liu Huimin Liu Jianxing Cai Jianxing Cai Jianxing Cai Lu Meng Lei Dong Nan Zheng Jiaqi Wang Cheng Wang Cheng Wang |
| author_sort | Yankun Zhao |
| collection | DOAJ |
| description | Streptococcus agalactiae is an important pathogen causing bovine mastitis. The aim of this study was to develop a simple and specific method for direct detection of S. agalactiae from milk products. Propidium monoazide (PMA) and sodium dodecyl sulfate (SDS) were utilized to eliminate the interference of dead and injured cells in qPCR. Lysozyme (LYZ) was adopted to increase the extraction efficiency of target bacteria DNA in milk matrix. The specific primers were designed based on cfb gene of S. agalactiae for qPCR. The inclusivity and exclusivity of the assay were evaluated using 30 strains. The method was further determined by the detection of S. agalactiae in spiked milk. Results showed significant differences between the SDS–PMA–qPCR, PMA–qPCR and qPCR when a final concentration of 10 mg/ml (R2 = 0.9996, E = 95%) of LYZ was added in DNA extraction. Viable S. agalactiae was effectively detected when SDS and PMA concentrations were 20 μg/ml and 10 μM, respectively, and it was specific and more sensitive than qPCR and PMA–qPCR. Moreover, the SDS–PMA–qPCR assay coupled with LYZ was used to detect viable S. agalactiae in spiked milk, with a limit of detection of 3 × 103 cfu/ml. Therefore, the SDS–PMA–qPCR assay had excellent sensitivity and specificity for detection of viable S. agalactiae in milk. |
| first_indexed | 2024-12-15T00:35:14Z |
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| id | doaj.art-8e962a95f40b43cf978dd749b1132ce4 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-15T00:35:14Z |
| publishDate | 2019-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-8e962a95f40b43cf978dd749b1132ce42022-12-21T22:41:50ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2019-03-011010.3389/fmicb.2019.00661441858Quantitative Polymerase Chain Reaction Coupled With Sodium Dodecyl Sulfate and Propidium Monoazide for Detection of Viable Streptococcus agalactiae in MilkYankun Zhao0Yankun Zhao1Yankun Zhao2He Chen3He Chen4He Chen5Huimin Liu6Huimin Liu7Huimin Liu8Huimin Liu9Jianxing Cai10Jianxing Cai11Jianxing Cai12Lu Meng13Lei Dong14Nan Zheng15Jiaqi Wang16Cheng Wang17Cheng Wang18Institute of Quality Standard and Testing Technology for Agro-Products, Xinjiang Academy of Agricultural Sciences, Urumqi, ChinaMinistry of Agriculture and Rural Affairs-Laboratory of Quality and Safety Risk Assessment for Agro-Products, Urumqi, ChinaKey Laboratory of Agro-Products Quality and Safety of Xinjiang, Urumqi, ChinaInstitute of Quality Standard and Testing Technology for Agro-Products, Xinjiang Academy of Agricultural Sciences, Urumqi, ChinaMinistry of Agriculture and Rural Affairs-Laboratory of Quality and Safety Risk Assessment for Agro-Products, Urumqi, ChinaKey Laboratory of Agro-Products Quality and Safety of Xinjiang, Urumqi, ChinaInstitute of Quality Standard and Testing Technology for Agro-Products, Xinjiang Academy of Agricultural Sciences, Urumqi, ChinaMinistry of Agriculture and Rural Affairs-Laboratory of Quality and Safety Risk Assessment for Agro-Products, Urumqi, ChinaKey Laboratory of Agro-Products Quality and Safety of Xinjiang, Urumqi, ChinaMinistry of Agriculture Laboratory of Quality and Safety Risk Assessment for Dairy Products (Beijing), Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, ChinaInstitute of Quality Standard and Testing Technology for Agro-Products, Xinjiang Academy of Agricultural Sciences, Urumqi, ChinaMinistry of Agriculture and Rural Affairs-Laboratory of Quality and Safety Risk Assessment for Agro-Products, Urumqi, ChinaKey Laboratory of Agro-Products Quality and Safety of Xinjiang, Urumqi, ChinaMinistry of Agriculture Laboratory of Quality and Safety Risk Assessment for Dairy Products (Beijing), Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, ChinaMinistry of Agriculture Laboratory of Quality and Safety Risk Assessment for Dairy Products (Beijing), Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, ChinaMinistry of Agriculture Laboratory of Quality and Safety Risk Assessment for Dairy Products (Beijing), Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, ChinaMinistry of Agriculture Laboratory of Quality and Safety Risk Assessment for Dairy Products (Beijing), Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, ChinaMinistry of Agriculture and Rural Affairs-Laboratory of Quality and Safety Risk Assessment for Agro-Products, Urumqi, ChinaKey Laboratory of Agro-Products Quality and Safety of Xinjiang, Urumqi, ChinaStreptococcus agalactiae is an important pathogen causing bovine mastitis. The aim of this study was to develop a simple and specific method for direct detection of S. agalactiae from milk products. Propidium monoazide (PMA) and sodium dodecyl sulfate (SDS) were utilized to eliminate the interference of dead and injured cells in qPCR. Lysozyme (LYZ) was adopted to increase the extraction efficiency of target bacteria DNA in milk matrix. The specific primers were designed based on cfb gene of S. agalactiae for qPCR. The inclusivity and exclusivity of the assay were evaluated using 30 strains. The method was further determined by the detection of S. agalactiae in spiked milk. Results showed significant differences between the SDS–PMA–qPCR, PMA–qPCR and qPCR when a final concentration of 10 mg/ml (R2 = 0.9996, E = 95%) of LYZ was added in DNA extraction. Viable S. agalactiae was effectively detected when SDS and PMA concentrations were 20 μg/ml and 10 μM, respectively, and it was specific and more sensitive than qPCR and PMA–qPCR. Moreover, the SDS–PMA–qPCR assay coupled with LYZ was used to detect viable S. agalactiae in spiked milk, with a limit of detection of 3 × 103 cfu/ml. Therefore, the SDS–PMA–qPCR assay had excellent sensitivity and specificity for detection of viable S. agalactiae in milk.https://www.frontiersin.org/article/10.3389/fmicb.2019.00661/fullStreptococcus agalactiaepropidium monoazidesodium dodecyl sulfateqPCRmilk |
| spellingShingle | Yankun Zhao Yankun Zhao Yankun Zhao He Chen He Chen He Chen Huimin Liu Huimin Liu Huimin Liu Huimin Liu Jianxing Cai Jianxing Cai Jianxing Cai Lu Meng Lei Dong Nan Zheng Jiaqi Wang Cheng Wang Cheng Wang Quantitative Polymerase Chain Reaction Coupled With Sodium Dodecyl Sulfate and Propidium Monoazide for Detection of Viable Streptococcus agalactiae in Milk Frontiers in Microbiology Streptococcus agalactiae propidium monoazide sodium dodecyl sulfate qPCR milk |
| title | Quantitative Polymerase Chain Reaction Coupled With Sodium Dodecyl Sulfate and Propidium Monoazide for Detection of Viable Streptococcus agalactiae in Milk |
| title_full | Quantitative Polymerase Chain Reaction Coupled With Sodium Dodecyl Sulfate and Propidium Monoazide for Detection of Viable Streptococcus agalactiae in Milk |
| title_fullStr | Quantitative Polymerase Chain Reaction Coupled With Sodium Dodecyl Sulfate and Propidium Monoazide for Detection of Viable Streptococcus agalactiae in Milk |
| title_full_unstemmed | Quantitative Polymerase Chain Reaction Coupled With Sodium Dodecyl Sulfate and Propidium Monoazide for Detection of Viable Streptococcus agalactiae in Milk |
| title_short | Quantitative Polymerase Chain Reaction Coupled With Sodium Dodecyl Sulfate and Propidium Monoazide for Detection of Viable Streptococcus agalactiae in Milk |
| title_sort | quantitative polymerase chain reaction coupled with sodium dodecyl sulfate and propidium monoazide for detection of viable streptococcus agalactiae in milk |
| topic | Streptococcus agalactiae propidium monoazide sodium dodecyl sulfate qPCR milk |
| url | https://www.frontiersin.org/article/10.3389/fmicb.2019.00661/full |
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