| Staphylococcus aureus(S.aureus)is an important zoonotic pathogen,as well as an important food-borne pathogen.The harm caused by this pathogen is one of the major public health problems of global concern.The rapid and accurate detection of this pathogen is an important part of the prevention and control of its harmful effects.Therefore,it is urgent to develop reliable and efficient technologies to detect S.aureus,so as to ensure the prevention and control of infectious diseases and reduce the occurrence of food poisoning incidents caused by this pathogen.Lysin is a kind of cell wall hydrolase encoded by dsDNA phage,which plays an important role in the later period of bacteriophage infection.Due to its high specificity and affinity for host bacteria,lysin has good application potential as a novel sensing element for the detection of various pathogens.A virulent phage GH15 that can specifically infect S.aureus was isolated and its lysin LysGH15 was successfully expressed.LysGH15 has a broad spectrum and high efficiency activity against S.aureus.Therefore,in this paper,LysGH15-C54A,a mutant lysin that retains only binding activity but no lysis activity for S.aureus,was expressed as a recognition element for S.aureus,and new methods for rapid,efficient and accurate detection of S.aureus was established.The established methods were also evaluated and preliminarily applied.Study I:A new detection method of S.aureus was established by combining LysGH15 and biolayer interferometry(BLI)technology.Firstly,LysGH15-C54A and the lyase binding domain SH3b(LysGH15B-GFP)were immobilized respectively on the probes of BLI sensor by amino coupling,and the detection of S.aureus was realized by the specificity of host bacteria recognition.Through the comparison of signal values,it was concluded that LysGH15-C54A has a better detection effect,so LysGH15-C54A was used for subsequent detection.By optimizing the detection conditions,the limit of detection of S.aureus were 151,38,18 and 13 CFU/mL when the binding time was 3,6,9 and 12 min,respectively.The method is specific enough to exclude potential interference from six other genera of bacteria:Escherichia coli,Yersinia enterocolitica,Pseudomonas aeruginosa,Listeria monocytogenes,Bacillus subtilis and Salmonella enteritidis.In addition,by adding the Native-LysGH15 for secondary combination,the viable and dead S.aureus can be distinguished.This method has been successfully applied to the detection of S.aureus in ice cubes and soy sauce,and the recovery rate at different times was above 98.86%,and the relative standard deviation were all below7.77%.These results demonstrate that the BLI assay based on the lysin LysGH15 holds promise as a novel diagnostic tool for S.aureus detection,indicating that LysGH15 has good application as the detection element of S.aureus.In study II,a colloidal gold immunochromatography assay(GICA)based on LysGH15-C54A was established.Based on Study I,LysGH15-C54A was fixed on the colloidal gold test strip as a recognition molecule in the detection region.At the same time,colloidal gold labeled with commercial polyclonal antibody of S.aureus was used as a probe to form a complex with S.aureus in advance.The LysGH15-C54A was used to capture the complex on the test line to deposit colloidal gold particles and generate visual bands for the detection of S.aureus.After optimizing the detection conditions,the detection sensitivity of the test strip was 5.0×10~2 CFU/mL.The interference of non-target bacteria such as Escherichia coli and Pseudomonas aeruginosa can be ruled out,but other strains of Staphylococcus such as Staphylococcus saprophyticus and Staphylococcus epidermidis also showed positive results.In view of the insufficiently low detection limit compared with BLI method and the interference of other Staphylococcus strains,further optimization of the detection method is required.In Study III,a new method for the rapid detection of S.aureus by combining immunomagnetic bead enrichment and colloidal gold test strips was established.On the basis of the above study,by increasing the method of immunomagnetic separation method and using chicken anti-protein IgY functionalized carboxyl magnetic beads,not only S.aureus can be successfully isolated in complex matrix,but also the potential interference of other strains of Staphylococcus can be eliminated.Then,the bacteria separated from immunomagnetic beads were detected by test strips,and a S.aureus detection method combining IgY-based immunomagnetic separation technology with LysGH15-C54A-based GICA was established(IMBs-GICA).The method can successfully detect 40 CFU/mL of S.aureus within 35 min.The dual recognition mode ensures the specificity of the established detection method,and can eliminate the interference of Staphylococcus saprophyticus,Staphylococcus epidermidis,and eight other genera of bacteria.The IMBs-GICA method has also been successfully used for the detection of S.aureus in freshly collected milk(200)and sputum(185),urine(107),bronchoalveolar lavage fluid(74),and serum(60)of clinical samples.The sensitivity and specificity were 92.3%and 98.9%for milk samples and 89.5%and 99.5%for clinical specimens overall.By comparing,this method was found to be superior to the traditional PCR method,with a detection rate closer to that of the assay gold standard culture method,while requiring less time.To sum up,we used the phage lysin LysGH15-C54A as a capture element of S.aureus and combined with BLI technology and GICA to established rapid,sensitive and specific detection methods by using the specific binding ability of LysGH15-C54A to S.aureus.All of the above methods are the first time to apply the complete phage lysin to the corresponding technology,shortening the detection time of S.aureus while maintaining a high sensitivity.In addition,these methods have successfully detected S.aureus in actual samples of veterinary,medical and food samples,which is expected to provide technical means for the diagnosis and prevention of S.aureus and has a good prospect in practical application. |
