Identification and quantification of viable Lacticaseibacillus rhamnosus in probiotics using validated PMA-qPCR method
The identification and quantification of viable bacteria at the species/strain level in compound probiotic products is challenging now. Molecular biology methods, e.g., propidium monoazide (PMA) combination with qPCR, have gained prominence for targeted viable cell counts. This study endeavors to es...
| প্রধান লেখক: | , , , , , , , , , , |
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| বিন্যাস: | প্রবন্ধ |
| ভাষা: | English |
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Frontiers Media S.A.
2024-01-01
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| মালা: | Frontiers in Microbiology |
| বিষয়গুলি: | |
| অনলাইন ব্যবহার করুন: | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1341884/full |
| _version_ | 1827380437643689984 |
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| author | Lizheng Guo Xiaolei Ze Huifen Feng Yiru Liu Yuanyuan Ge Xi Zhao Chengyu Song Yingxin Jiao Jiaqi Liu Shuaicheng Mu Su Yao |
| author_facet | Lizheng Guo Xiaolei Ze Huifen Feng Yiru Liu Yuanyuan Ge Xi Zhao Chengyu Song Yingxin Jiao Jiaqi Liu Shuaicheng Mu Su Yao |
| author_sort | Lizheng Guo |
| collection | DOAJ |
| description | The identification and quantification of viable bacteria at the species/strain level in compound probiotic products is challenging now. Molecular biology methods, e.g., propidium monoazide (PMA) combination with qPCR, have gained prominence for targeted viable cell counts. This study endeavors to establish a robust PMA-qPCR method for viable Lacticaseibacillus rhamnosus detection and systematically validated key metrics encompassing relative trueness, accuracy, limit of quantification, linear, and range. The inclusivity and exclusivity notably underscored high specificity of the primers for L. rhamnosus, which allowed accurate identification of the target bacteria. Furthermore, the conditions employed for PMA treatment were fully verified by 24 different L. rhamnosus including type strain, commercial strains, etc., confirming its effective discrimination between live and dead bacteria. A standard curve constructed by type strain could apply to commercial strains to convert qPCR Cq values to viable cell numbers. The established PMA-qPCR method was applied to 46 samples including pure cultures, probiotics as food ingredients, and compound probiotic products. Noteworthy is the congruity observed between measured and theoretical values within a 95% confidence interval of the upper and lower limits of agreement, demonstrating the relative trueness of this method. Moreover, accurate results were obtained when viable L. rhamnosus ranging from 103 to 108 CFU/mL. The comprehensive appraisal of PMA-qPCR performances provides potential industrial applications of this new technology in quality control and supervision of probiotic products. |
| first_indexed | 2024-03-08T13:32:52Z |
| format | Article |
| id | doaj.art-d8b9da0345fc407ea6ec4e7b4b659701 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-03-08T13:32:52Z |
| publishDate | 2024-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-d8b9da0345fc407ea6ec4e7b4b6597012024-01-17T04:43:07ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2024-01-011510.3389/fmicb.2024.13418841341884Identification and quantification of viable Lacticaseibacillus rhamnosus in probiotics using validated PMA-qPCR methodLizheng Guo0Xiaolei Ze1Huifen Feng2Yiru Liu3Yuanyuan Ge4Xi Zhao5Chengyu Song6Yingxin Jiao7Jiaqi Liu8Shuaicheng Mu9Su Yao10China National Research Institute of Food and Fermentation Industries Co., LTD., China Center of Industrial Culture Collection, Beijing, ChinaMicrobiome Research and Application Center, BYHEALTH Institute of Nutrition & Health, Guangzhou, ChinaChina National Research Institute of Food and Fermentation Industries Co., LTD., China Center of Industrial Culture Collection, Beijing, ChinaChina National Research Institute of Food and Fermentation Industries Co., LTD., China Center of Industrial Culture Collection, Beijing, ChinaChina National Research Institute of Food and Fermentation Industries Co., LTD., China Center of Industrial Culture Collection, Beijing, ChinaMicrobiome Research and Application Center, BYHEALTH Institute of Nutrition & Health, Guangzhou, ChinaChina National Research Institute of Food and Fermentation Industries Co., LTD., China Center of Industrial Culture Collection, Beijing, ChinaChina National Research Institute of Food and Fermentation Industries Co., LTD., China Center of Industrial Culture Collection, Beijing, ChinaChina National Research Institute of Food and Fermentation Industries Co., LTD., China Center of Industrial Culture Collection, Beijing, ChinaChina National Research Institute of Food and Fermentation Industries Co., LTD., China Center of Industrial Culture Collection, Beijing, ChinaChina National Research Institute of Food and Fermentation Industries Co., LTD., China Center of Industrial Culture Collection, Beijing, ChinaThe identification and quantification of viable bacteria at the species/strain level in compound probiotic products is challenging now. Molecular biology methods, e.g., propidium monoazide (PMA) combination with qPCR, have gained prominence for targeted viable cell counts. This study endeavors to establish a robust PMA-qPCR method for viable Lacticaseibacillus rhamnosus detection and systematically validated key metrics encompassing relative trueness, accuracy, limit of quantification, linear, and range. The inclusivity and exclusivity notably underscored high specificity of the primers for L. rhamnosus, which allowed accurate identification of the target bacteria. Furthermore, the conditions employed for PMA treatment were fully verified by 24 different L. rhamnosus including type strain, commercial strains, etc., confirming its effective discrimination between live and dead bacteria. A standard curve constructed by type strain could apply to commercial strains to convert qPCR Cq values to viable cell numbers. The established PMA-qPCR method was applied to 46 samples including pure cultures, probiotics as food ingredients, and compound probiotic products. Noteworthy is the congruity observed between measured and theoretical values within a 95% confidence interval of the upper and lower limits of agreement, demonstrating the relative trueness of this method. Moreover, accurate results were obtained when viable L. rhamnosus ranging from 103 to 108 CFU/mL. The comprehensive appraisal of PMA-qPCR performances provides potential industrial applications of this new technology in quality control and supervision of probiotic products.https://www.frontiersin.org/articles/10.3389/fmicb.2024.1341884/fullprobioticsLacticaseibacillus rhamnosusidentificationviable cell quantificationPMA-qPCR methodvalidation |
| spellingShingle | Lizheng Guo Xiaolei Ze Huifen Feng Yiru Liu Yuanyuan Ge Xi Zhao Chengyu Song Yingxin Jiao Jiaqi Liu Shuaicheng Mu Su Yao Identification and quantification of viable Lacticaseibacillus rhamnosus in probiotics using validated PMA-qPCR method Frontiers in Microbiology probiotics Lacticaseibacillus rhamnosus identification viable cell quantification PMA-qPCR method validation |
| title | Identification and quantification of viable Lacticaseibacillus rhamnosus in probiotics using validated PMA-qPCR method |
| title_full | Identification and quantification of viable Lacticaseibacillus rhamnosus in probiotics using validated PMA-qPCR method |
| title_fullStr | Identification and quantification of viable Lacticaseibacillus rhamnosus in probiotics using validated PMA-qPCR method |
| title_full_unstemmed | Identification and quantification of viable Lacticaseibacillus rhamnosus in probiotics using validated PMA-qPCR method |
| title_short | Identification and quantification of viable Lacticaseibacillus rhamnosus in probiotics using validated PMA-qPCR method |
| title_sort | identification and quantification of viable lacticaseibacillus rhamnosus in probiotics using validated pma qpcr method |
| topic | probiotics Lacticaseibacillus rhamnosus identification viable cell quantification PMA-qPCR method validation |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1341884/full |
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