Evaluation of a direct phage DNA detection-based Taqman qPCR methodology for quantification of phage and its application in rapid ultrasensitive identification of Acinetobacter baumannii
Abstract Background Rapid phage enumeration/quantitation and viable bacteria determination is critical for phage application and treatment of infectious patients caused by the pathogenic bacteria. Methods In the current study, a direct phage DNA detection-based Taqman qPCR methodology for quantifica...
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| Format: | Article |
| Language: | English |
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BMC
2022-06-01
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| Series: | BMC Infectious Diseases |
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| Online Access: | https://doi.org/10.1186/s12879-022-07493-1 |
| _version_ | 1818211662289698816 |
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| author | Jun Luo Min Liu Peng Wang Qianyuan Li Chunhua Luo Hongping Wei Yuanyuan Hu Junping Yu |
| author_facet | Jun Luo Min Liu Peng Wang Qianyuan Li Chunhua Luo Hongping Wei Yuanyuan Hu Junping Yu |
| author_sort | Jun Luo |
| collection | DOAJ |
| description | Abstract Background Rapid phage enumeration/quantitation and viable bacteria determination is critical for phage application and treatment of infectious patients caused by the pathogenic bacteria. Methods In the current study, a direct phage DNA detection-based Taqman qPCR methodology for quantification of phage P53 and rapid ultrasensitive identification of Acinetobacter baumannii (A. baumannii) was evaluated. Results The assay was capable of quantifying P53 phage DNA without DNA extraction and the detection limit of the assay was 550 PFU/mL. The agreement bias between the quantitative results of three different phage concentrations in this assay and double agar overlay plaque assay were under 3.38%. Through the built detection system, down to 1 log CFU/mL of viable A. baumannii can be detected within 4 h in A. baumannii spiked swab and bronchoalveolar lavage fluid samples. Compared with the Taqman qPCR that targets the conserved sequence of A. baumannii, the sensitivity of the assay built in this study could increase four orders of magnitude. Conclusions The methodology offers a valid alternative for enumeration of freshly prepared phage solution and diagnosis of bacterial infection caused by A. baumannii or other bacterial infection in complicated samples through switching to phages against other bacteria. Furthermore, the assay could offer drug adjustment strategy timely owing to the detection of bacteria vitality. |
| first_indexed | 2024-12-12T05:36:04Z |
| format | Article |
| id | doaj.art-98290556836e40899fa8ae23c38e1f19 |
| institution | Directory Open Access Journal |
| issn | 1471-2334 |
| language | English |
| last_indexed | 2024-12-12T05:36:04Z |
| publishDate | 2022-06-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Infectious Diseases |
| spelling | doaj.art-98290556836e40899fa8ae23c38e1f192022-12-22T00:36:09ZengBMCBMC Infectious Diseases1471-23342022-06-012211910.1186/s12879-022-07493-1Evaluation of a direct phage DNA detection-based Taqman qPCR methodology for quantification of phage and its application in rapid ultrasensitive identification of Acinetobacter baumanniiJun Luo0Min Liu1Peng Wang2Qianyuan Li3Chunhua Luo4Hongping Wei5Yuanyuan Hu6Junping Yu7The First College of Clinical Medical Science, China Three Gorges UniversityThe First College of Clinical Medical Science, China Three Gorges UniversityThe First College of Clinical Medical Science, China Three Gorges UniversityThe First College of Clinical Medical Science, China Three Gorges UniversityThe First College of Clinical Medical Science, China Three Gorges UniversityCAS Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of SciencesMedical College, China Three Gorges UniversityCAS Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of SciencesAbstract Background Rapid phage enumeration/quantitation and viable bacteria determination is critical for phage application and treatment of infectious patients caused by the pathogenic bacteria. Methods In the current study, a direct phage DNA detection-based Taqman qPCR methodology for quantification of phage P53 and rapid ultrasensitive identification of Acinetobacter baumannii (A. baumannii) was evaluated. Results The assay was capable of quantifying P53 phage DNA without DNA extraction and the detection limit of the assay was 550 PFU/mL. The agreement bias between the quantitative results of three different phage concentrations in this assay and double agar overlay plaque assay were under 3.38%. Through the built detection system, down to 1 log CFU/mL of viable A. baumannii can be detected within 4 h in A. baumannii spiked swab and bronchoalveolar lavage fluid samples. Compared with the Taqman qPCR that targets the conserved sequence of A. baumannii, the sensitivity of the assay built in this study could increase four orders of magnitude. Conclusions The methodology offers a valid alternative for enumeration of freshly prepared phage solution and diagnosis of bacterial infection caused by A. baumannii or other bacterial infection in complicated samples through switching to phages against other bacteria. Furthermore, the assay could offer drug adjustment strategy timely owing to the detection of bacteria vitality.https://doi.org/10.1186/s12879-022-07493-1Direct phage DNA detection-based Taqman qPCRQuantification of phageViable Acinetobacter baumanniiDetectionSwab/bronchoalveolar lavage fluid |
| spellingShingle | Jun Luo Min Liu Peng Wang Qianyuan Li Chunhua Luo Hongping Wei Yuanyuan Hu Junping Yu Evaluation of a direct phage DNA detection-based Taqman qPCR methodology for quantification of phage and its application in rapid ultrasensitive identification of Acinetobacter baumannii BMC Infectious Diseases Direct phage DNA detection-based Taqman qPCR Quantification of phage Viable Acinetobacter baumannii Detection Swab/bronchoalveolar lavage fluid |
| title | Evaluation of a direct phage DNA detection-based Taqman qPCR methodology for quantification of phage and its application in rapid ultrasensitive identification of Acinetobacter baumannii |
| title_full | Evaluation of a direct phage DNA detection-based Taqman qPCR methodology for quantification of phage and its application in rapid ultrasensitive identification of Acinetobacter baumannii |
| title_fullStr | Evaluation of a direct phage DNA detection-based Taqman qPCR methodology for quantification of phage and its application in rapid ultrasensitive identification of Acinetobacter baumannii |
| title_full_unstemmed | Evaluation of a direct phage DNA detection-based Taqman qPCR methodology for quantification of phage and its application in rapid ultrasensitive identification of Acinetobacter baumannii |
| title_short | Evaluation of a direct phage DNA detection-based Taqman qPCR methodology for quantification of phage and its application in rapid ultrasensitive identification of Acinetobacter baumannii |
| title_sort | evaluation of a direct phage dna detection based taqman qpcr methodology for quantification of phage and its application in rapid ultrasensitive identification of acinetobacter baumannii |
| topic | Direct phage DNA detection-based Taqman qPCR Quantification of phage Viable Acinetobacter baumannii Detection Swab/bronchoalveolar lavage fluid |
| url | https://doi.org/10.1186/s12879-022-07493-1 |
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