Development of Multiplex qPCR Method for Accurate Detection of Enzyme-Producing Psychrotrophic Bacteria
Microbial detection in milk is crucial for food safety and quality, as beneficial and harmful microorganisms can affect consumer health and dairy product integrity. Identifying and quantifying these microorganisms helps prevent contamination and spoilage. The study employs advanced molecular techniq...
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2025-06-01
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| author | Kidane Yalew Shuwen Zhang Solomon Gebreyowhans Ning Xie Yunna Wang Jiaping Lv Xu Li Xiaoyang Pang |
| author_facet | Kidane Yalew Shuwen Zhang Solomon Gebreyowhans Ning Xie Yunna Wang Jiaping Lv Xu Li Xiaoyang Pang |
| author_sort | Kidane Yalew |
| collection | DOAJ |
| description | Microbial detection in milk is crucial for food safety and quality, as beneficial and harmful microorganisms can affect consumer health and dairy product integrity. Identifying and quantifying these microorganisms helps prevent contamination and spoilage. The study employs advanced molecular techniques to detect and quantify the genomic DNA for the target hydrolytic enzyme coding genes <i>lipA</i> and <i>aprX</i> based on the multi-align sequence conserved region, specific primer pair, and hydrolysis probes designed using the singleplex qPCR and multiplex qPCR. Cultured isolates and artificially contaminated sterilized ultra-high-temperature (UHT) milk were analyzed for their specificity, cross-reactivity, and sensitivity. The finding indicated that strains with <i>lipA</i> and <i>aprX</i> genes were amplified while the other strains were not amplified. This indicated that the designed primer pairs/probes were very specific to the target gene of interest. The specificity of each design primer pair was checked using SYBR Green qPCR using 16 different isolate strains from the milk sample. The quantification specificity of each strain target gene was deemed to be with a mean Ct value for positive pseudomonas strain > 16.98 ± 1.76 (<i>p</i> < 0.0001), non-pseudomonas positive strain ≥ 27.47 ± 1.25 (<i>p</i> < 0.0001), no Ct for the negative control and molecular grade water. The sensitivity limit of detection (LOD) analyzed based on culture broth and milk sample was >10<sup>5</sup> and >10<sup>4</sup> in PCR amplification while it was >10<sup>4</sup> and >10<sup>3</sup> in real-time qPCR, respectively. At the same time, the correlation regression coefficient of the standard curve based on the pure culture cell DNA as the DNA concentration serially diluted (20 ng/µL to 0.0002 ng/µL) was obtained in multiplex without interference and cross-reactivity, yielding R<sup>2</sup> ≥ 0.9908 slope (−3.2591) and intercepting with a value of 37, where the efficiency reached the level of 95–102% sensitivity reached up to 0.0002 ng/µL concentration of DNA, and sensitivity of microbial load was up to 1.2 × 10<sup>2</sup> CFU/mL. Therefore, multiplex TaqMan qPCR simultaneous amplification was considered the best method developed for the detection of the <i>lipA</i> and <i>aprX</i> genes in a single tube. This will result in developing future simultaneous (three- to four-gene) detection of spoilage psychrotrophic bacteria in raw milk. |
| format | Article |
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| institution | OA Journals |
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| language | English |
| publishDate | 2025-06-01 |
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| spelling | doaj-art-44d7785fb4b14e3883b3c700be36617e2025-08-20T02:23:07ZengMDPI AGFoods2304-81582025-06-011411197510.3390/foods14111975Development of Multiplex qPCR Method for Accurate Detection of Enzyme-Producing Psychrotrophic BacteriaKidane Yalew0Shuwen Zhang1Solomon Gebreyowhans2Ning Xie3Yunna Wang4Jiaping Lv5Xu Li6Xiaoyang Pang7Key Laboratory of Agro-products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs, Institution of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaKey Laboratory of Agro-products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs, Institution of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaTigray Agricultural Research Institution, Mekelle 0492, EthiopiaKey Laboratory of Agro-products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs, Institution of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaKey Laboratory of Agro-products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs, Institution of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaKey Laboratory of Agro-products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs, Institution of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaKey Laboratory of Agro-products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs, Institution of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaKey Laboratory of Agro-products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs, Institution of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaMicrobial detection in milk is crucial for food safety and quality, as beneficial and harmful microorganisms can affect consumer health and dairy product integrity. Identifying and quantifying these microorganisms helps prevent contamination and spoilage. The study employs advanced molecular techniques to detect and quantify the genomic DNA for the target hydrolytic enzyme coding genes <i>lipA</i> and <i>aprX</i> based on the multi-align sequence conserved region, specific primer pair, and hydrolysis probes designed using the singleplex qPCR and multiplex qPCR. Cultured isolates and artificially contaminated sterilized ultra-high-temperature (UHT) milk were analyzed for their specificity, cross-reactivity, and sensitivity. The finding indicated that strains with <i>lipA</i> and <i>aprX</i> genes were amplified while the other strains were not amplified. This indicated that the designed primer pairs/probes were very specific to the target gene of interest. The specificity of each design primer pair was checked using SYBR Green qPCR using 16 different isolate strains from the milk sample. The quantification specificity of each strain target gene was deemed to be with a mean Ct value for positive pseudomonas strain > 16.98 ± 1.76 (<i>p</i> < 0.0001), non-pseudomonas positive strain ≥ 27.47 ± 1.25 (<i>p</i> < 0.0001), no Ct for the negative control and molecular grade water. The sensitivity limit of detection (LOD) analyzed based on culture broth and milk sample was >10<sup>5</sup> and >10<sup>4</sup> in PCR amplification while it was >10<sup>4</sup> and >10<sup>3</sup> in real-time qPCR, respectively. At the same time, the correlation regression coefficient of the standard curve based on the pure culture cell DNA as the DNA concentration serially diluted (20 ng/µL to 0.0002 ng/µL) was obtained in multiplex without interference and cross-reactivity, yielding R<sup>2</sup> ≥ 0.9908 slope (−3.2591) and intercepting with a value of 37, where the efficiency reached the level of 95–102% sensitivity reached up to 0.0002 ng/µL concentration of DNA, and sensitivity of microbial load was up to 1.2 × 10<sup>2</sup> CFU/mL. Therefore, multiplex TaqMan qPCR simultaneous amplification was considered the best method developed for the detection of the <i>lipA</i> and <i>aprX</i> genes in a single tube. This will result in developing future simultaneous (three- to four-gene) detection of spoilage psychrotrophic bacteria in raw milk.https://www.mdpi.com/2304-8158/14/11/1975hydrolytic enzymesmicrobial detectionmultiplex qPCRpsychrotrophic bacteriaraw milk |
| spellingShingle | Kidane Yalew Shuwen Zhang Solomon Gebreyowhans Ning Xie Yunna Wang Jiaping Lv Xu Li Xiaoyang Pang Development of Multiplex qPCR Method for Accurate Detection of Enzyme-Producing Psychrotrophic Bacteria Foods hydrolytic enzymes microbial detection multiplex qPCR psychrotrophic bacteria raw milk |
| title | Development of Multiplex qPCR Method for Accurate Detection of Enzyme-Producing Psychrotrophic Bacteria |
| title_full | Development of Multiplex qPCR Method for Accurate Detection of Enzyme-Producing Psychrotrophic Bacteria |
| title_fullStr | Development of Multiplex qPCR Method for Accurate Detection of Enzyme-Producing Psychrotrophic Bacteria |
| title_full_unstemmed | Development of Multiplex qPCR Method for Accurate Detection of Enzyme-Producing Psychrotrophic Bacteria |
| title_short | Development of Multiplex qPCR Method for Accurate Detection of Enzyme-Producing Psychrotrophic Bacteria |
| title_sort | development of multiplex qpcr method for accurate detection of enzyme producing psychrotrophic bacteria |
| topic | hydrolytic enzymes microbial detection multiplex qPCR psychrotrophic bacteria raw milk |
| url | https://www.mdpi.com/2304-8158/14/11/1975 |
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