Establishment Of RT-qsCPA And CPAP Visual Methods For Rapid Detection Of Vibrio Parahaemolyticus

Vibrio parahaemolyticus,belonging to Vibrio genus,is a foodborne pathogen that is widely distributed in nature.If people eat food contaminated with Vibrio parahaemolyticus,it could cause acute gastroenteritis with abdominal pain,diarrhea,nausea,vomiting and fever.In addition,severe cases can also occur dehydration,shock coma and even death,so Vibrio parahaemolyticus is also known as Vibrio‘enteritise'.Vibrio parahaemolyticus is liable to cause serious pollution of fresh foods because of its rapidly growth and proliferation at 26?leading to the amount increase by 50⁷90 times within 24 hours.At present,Vibrio parahaemolyticus has become the main food-borne pathogen in China,especially in some coastal cities.The cases caused by Vibrio parahaemolyticus account for over 60%among the total number of bacterial food poisoning cases.However,the traditional method for Vibrio parahaemolyticus detection is operating cumbersome,time consuming and heavy workload.Therefore,it is of great significance for monitoring food safety to establish a rapid,convenient and sensitive detection method.At present,cross-primer amplification?CPA?mainly combines nano-gold test strips to detect targets,and other detection methods derived from CPA technology are still very few,far less than the development of LAMP technology.In order to further developing and applicating of CPA technology,this study based on CPA technology established real-time quantitative single cross-primering amplification?RT-qsCPA?and cross-primering amplification-HPO₄^2-?CPAP?methods for detecting Vibrio parahaemolyticus.In this study,the tlh of Vibrio parahaemolyticus was chosen as target gene to design CPA primers?backward inner primer 2a and 3a,cross primer,and a pair of outer primer 4s and 5a?.The composition and reaction conditions for RT-qsCPA detection method and CPAP visual detection method were optimized;investigating the sensitivity and specificity of the two methods;comparing the effects of different nanoparticles on CPA amplification efficiency to choose suitable nanoparticles for improving reaction efficiency;comparing CPAP visual detection method and detection effect of phosphate test strips.The assay results show that:1.The optimum reaction mixture of RT-qsCPA contains 0.5×SYBR Green?,1×isothermal Amp Buffer,0.5 mol·L^-11 betaine,6 mmol·L^-11 MgSO₄,0.8 mmol·L^-11 dNTPs,0.5?mol·L^-11 cross primer,0.4?mol·L^-11 primer 2a,0.4?mol·L^-11 primer 3a,0.05?mol·L^-11 primer4s,0.05?mol·L^-11 primer 5a,8 U Bst 2.0 DNA polymerase,respectively.The optimum reaction procedure of RT-qsCPA is at 63?for 1 hour.2.The optimum reaction mixture of CPAP visual detection method contains1×isothermal Amp Buffer,0.5 mol·L^-11 betaine,4 mmol·L^-11 MgSO₄,0.6 mmol·L^-11 dNTPs,0.5?mol·L^-11 cross primer,0.4?mol·L^-11 primer 2a,0.4?mol·L^-11 primer 3a,0.05?mol·L^-1primer 4s,0.05?mol·L^-11 primer 5a,8 U Bst 2.0 DNA polymerase and 0.05 U thermostable inorganic pyrophosphatase?PPase?.The optimum composition of the chromogenic reagent for CPAP includes 1.68 mmol·L^-11 ammonium molybdate,0.08 mmol·L^-11 potassium antimonyl tartrate,0.3mol·L^-11 H₂SO₄,0.8%ascorbic acid.The optimum reaction procedure of CPAP visual detection method is at 63?for 35 min.3.0.2 mg·mL^-11 SiO₂ can promote amplification effect of RT-qsCPA.4.The LODs of RT-qsCPA and CPAP visual detection method are 10³copies,respectively.5.Compared with the result of phosphate test strips,the CPAP visual inspection method has a better efficacy for phosphate detection.By our reaserch,a rapid,convenient and sensitive detection method based on CPA technology for quantitative and visualization has been established,which provides a new detection technology platform for rapid detection of Vibrio parahaemolyticus in food.