阅读说明：本技术 一种水体中副溶血弧菌的现场检测试剂盒和检测方法 (On-site detection kit and detection method for vibrio parahaemolyticus in water body ) 是由 曲克明 张旭志 朱建新 徐勇 张艳 杨倩倩 闫华 于 2020-08-05 设计创作，主要内容包括：本发明涉及一种水体中副溶血弧菌的现场检测试剂盒和检测方法,属于水质检测领域,所述试剂盒包括溴化N-十二烷基异喹啉试剂、副溶血弧菌烈性噬菌体悬液和不同浓度梯度的副溶血弧菌菌悬液。利用所述试剂盒进行检测水体中副溶血弧菌的方法为将待检测水样装在检测管中,加入专用烈性噬菌体,能够特异性、快速地裂解其中的副溶血弧菌,生成多种小分子和离子,从而引起σ数值增加。由于σ增量与给检测管所装水体中副溶血弧菌的数量成正比,所以测定Δσ即可计算出待测水体样品中副溶血弧菌的定量信息。该方法可以在养殖现场快速确定含有多种微生物的复杂水体中副溶血弧菌的量,为渔业生产管理提供高时效性、准确的支撑数据。(The invention relates to an on-site detection kit and a detection method for vibrio parahaemolyticus in a water body, belonging to the field of water quality detection. The method for detecting the vibrio parahaemolyticus in the water body by using the kit comprises the steps of filling a water sample to be detected in a detection tube, adding the special virulent phage, specifically and quickly cracking the vibrio parahaemolyticus in the water sample, and generating various small molecules and ions, so that the sigma value is increased. Because the sigma increment is in direct proportion to the quantity of the vibrio parahaemolyticus in the water body filled in the detection tube, the quantitative information of the vibrio parahaemolyticus in the water body sample to be detected can be calculated by measuring the delta sigma. The method can rapidly determine the amount of the vibrio parahaemolyticus in the complex water body containing various microorganisms on a culture site, and provides high timeliness and accurate support data for fishery production management.)

1. An on-site detection kit for vibrio parahaemolyticus in water, which is characterized by comprising a brominated N-dodecyl isoquinoline reagent, a vibrio parahaemolyticus virulent phage suspension and a concentration gradient of 10¹¹、10¹⁰、10⁹、10⁸、10⁷、10⁶And 10⁵CFU/ml of a suspension of Vibrio parahaemolyticus.

2. The method for detecting Vibrio parahaemolyticus in an aqueous body using the kit of claim 1, wherein the method comprises the steps of:

1) taking water to be measured, averagely dividing the water into two parts, and respectively filling the two parts into a container S_AAnd S_BTwo centrifuge tubes; wherein S_AAdding brominated N-dodecyl isoquinoline, performing ultrasonic treatment in a portable ultrasonic instrument for 5min at an ultrasonic power of 30W to completely crack all kinds of bacteria in the water body to prepare a negative control solution S_{Yin (kidney)}；

2) Taking a certain amount of S_{Yin (kidney)}Adding a suspension of Vibrio parahaemolyticus with known concentration to obtain a solution containing 10⁶Positive control solution S of CFU/ml vibrio parahaemolyticus_{Yang (Yang)}；

3) Taking S of the same volume_B、S_{Yin (kidney)}He Yang S_{Yang (Yang)}，S_BI.e. the sample S to be tested_MeasuringSeparately loading a detection tube T_Measuring、T_{Yin (kidney)}And T_{Yang (Yang)}Then adding equivalent vibrio parahaemolyticus virulent phage suspensions respectively and mixing uniformly;

4) will detect the tube T_Measuring、T_{Yin (kidney)}And T_{Yang (Yang)}Simultaneously and respectively putting the three detection channels into the chemical and biological process on-line monitoring system, and respectively recording the initial capacitive coupling non-contact conductance response values sigma of the detected liquid in the three detection tubes_{Measure beginning}、σ_{Beginning of yin}And σ_{Yang beginning}(ii) a After standing for 30min, respectively recording the end-point capacitance coupling non-contact conductance response values sigma of the measured liquid in the three detection tubes_{Test terminal}、σ_{Yin Final}And σ_{Yang Final}；

5) Respectively calculating S according to the following formula_Measuring、S_{Yin (kidney)}And S_{Yang (Yang)}The capacitance coupling non-contact conductivity response change value:

Δσ_measuring＝σ_{Test terminal}-σ_{Measure beginning}

Δσ_{Yin (kidney)}＝σ_{Yin Final}-σ_{Beginning of yin}

Δσ_{Yang (Yang)}＝σ_{Yang Final}-σ_{Yang beginning}

6) Will delta sigma_MeasuringSubstituting the numerical value into a working curve functional relation to obtain the concentration of the vibrio parahaemolyticus in the water body to be detected; according to σ_{Yin (kidney)}Less than or equal to 2 mu S/cm and delta sigma_{Yang (Yang)}The judgment experiment of more than or equal to 10 mu S/cm has no false positive and false negative.

3. The method according to claim 1, wherein the working curve of step 6) is formulated as follows:

1) according to the preparation of a positive control sample S_{Yang (Yang)}Respectively prepared by the same method as described above, respectively containing 10¹⁰、10⁹、10⁸、10⁷、10⁶、10⁵And 10⁴Bacterial suspension S of CFU/ml vibrio parahaemolyticus_{Yang 10}、S_{Yang 9}、S_{Yang 8}、S_{Yang 7}、S_{Yang 6}、S_{Yang 5}And S_{Yang 4}；

2) Take the same volume S_{Yang 10}、S_{Yang 9}、S_{Yang 8}、S_{Yang 7}、S_{Yang 6}、S_{Yang 5}、S_{Yang 4}And negative control S_{Yin (kidney)}Separately loaded into the detecting tubes T_{Yang 10}、T_{Yang 9}、T_{Yang 8}、T_{Yang 7}、T_{Yang 6}、T_{Yang 5}、T_{Yang 4}And T_{Yin (kidney)}Then adding equivalent vibrio parahaemolyticus virulent phage suspensions respectively and mixing uniformly;

3) will detect the tube T_{Yang 10}、T_{Yang 9}、T_{Yang 8}、T_{Yang 7}、T_{Yang 6}、T_{Yang 5}、T_{Yang 4}And T_{Yin (kidney)}Simultaneously and respectively inserting 8 detection tubes into 8 detection channels of the chemical and biological process on-line monitoring system, and respectively recording initial capacitive coupling non-contact conductance response values sigma of the detected liquid_{Yang 10 Yuan}、σ_{Yang 9 Yuan}、σ_{Yang 8 Yuan}、σ_{Yang 7 Yuan}、σ_{Yang 6 initial stage}、σ_{Yang 5 Yuan}、σ_{Yang 4 initial stage}And σ_{Beginning of yin}(ii) a After standing for 30min, respectively recording the end point capacitance coupling non-contact conductance response values sigma of the detected liquid in 8 detection tubes_{Yang 10 Final}、σ_{Yang 9 Final}、σ_{Yang 8 Final}、σ_{Yang 7 Final}、σ_{Yang 6 Final}、σ_{Yang 5 Final}、σ_{Yang 4 Final}And σ_{Yin Final}；

4) According to the formula delta sigma-sigma_{Final (a Chinese character of 'gan')}-σ_{First stage}Separately calculate S_{Yang 10}、S_{Yang 9}、S_{Yang 8}、S_{Yang 7}、S_{Yang 6}、S_{Yang 5}And S_{Yang 4}Initial response value sigma of capacitive coupling non-contact conductance_{First stage}And endpoint response value σ_{Final (a Chinese character of 'gan')}The difference between the two and plotting the obtained data on the concentration logC of the vibrio parahaemolyticus to obtain a functional relation between delta sigma and logC; at the same time, according to Δ σ_{Yin (kidney)}The test is judged to have no false positive result less than or equal to 2 mu S/cm.

The technical field is as follows:

the invention belongs to the field of water quality detection, and particularly relates to a field detection kit and a detection method for vibrio parahaemolyticus in a water body.

Background art:

vibrio parahaemolyticus (Vibrio parahaemolyticus) belongs to gram-negative bacteria, is halophilic, and widely exists in fishery environment and aquatic organisms, and a common method for detecting the Vibrio parahaemolyticus in a water body is mainly a national standard (GB4789.7-2013) method based on enrichment culture-isolated culture-biochemical reaction. Although the method is a gold standard, the method is complex in operation, high in labor intensity, long in period and high in accuracy, and the accuracy depends on the professional level of an operator, so that the on-site, rapid and accurate measurement cannot be realized. In recent years, molecular biology methods (Huangmenshi, Naja, Zhanyan, Valley sunlight, Zhao, Zhanxuzhi, Dingdongsheng, Qukeming. visual LAMP rapid detection method of vibrio parahaemolyticus in seawater, Chinese fishery quality and standard, 2017,7(6),58-65) and immunological detection methods (separation and identification, molecular typing of vibrio parahaemolyticus in seafood in Shanghai region, and cloning expression of tdh gene [ Master graduation paper ]; Nanjing agriculture university, 2013) based on gene detection have been rapidly developed, and detection efficiency of vibrio parahaemolyticus is significantly improved. However, the new method is limited by expensive instruments and professional analysis sites, and the on-site, rapid and accurate determination of vibrio parahaemolyticus cannot be realized at present. Therefore, effective early warning data cannot be provided for production activities such as aquaculture and the like.

The invention content is as follows:

the technical problem to be solved by the invention is to provide a field detection kit and a detection method for vibrio parahaemolyticus in water, and the working principle is as follows: a water sample to be detected is arranged in a detection tube and has a determined capacitance coupling non-contact conductance (sigma) value; the vibrio parahaemolyticus virulent phage is added, so that vibrio parahaemolyticus in the vibrio parahaemolyticus can be specifically and rapidly split, various small molecules and ions are generated, and the sigma value is increased. Because the sigma increment (delta sigma) is in direct proportion to the quantity of the vibrio parahaemolyticus in the water body filled in the detection tube, the quantitative information of the vibrio parahaemolyticus in the water body sample to be detected can be calculated by adopting a chemical and biological process on-line monitoring system to measure the delta sigma. The method can rapidly determine the amount of the vibrio parahaemolyticus in the complex water body containing various microorganisms on site, and provides high timeliness and accurate support data for fishery production management and other activities.

The invention is realized by the following technical scheme:

an on-site detection kit for vibrio parahaemolyticus in water comprises a brominated N-dodecyl isoquinoline reagent, a vibrio parahaemolyticus virulent phage suspension and a concentration gradient of 10¹¹、10¹⁰、10⁹、10⁸、10⁷、10⁶And 10⁵CFU/ml of a suspension of Vibrio parahaemolyticus.

The invention also provides a method for detecting vibrio parahaemolyticus in water by using the kit, which comprises the following steps:

1) taking water to be measured, averagely dividing the water into two parts, and respectively filling the two parts into a container S_AAnd S_BTwo centrifuge tubes; wherein S_AAdding brominated N-dodecyl isoquinoline, performing ultrasonic treatment in a portable ultrasonic instrument for 5min at an ultrasonic power of 30W to completely crack all kinds of bacteria in the water body to prepare a negative control solution S_{Yin (kidney)}；

2) Taking a certain amount of S_{Yin (kidney)}Adding a suspension of Vibrio parahaemolyticus with known concentration to obtain a solution containing 10⁶Positive control solution S of CFU/ml vibrio parahaemolyticus_{Yang (Yang)}；

3) Taking S of the same volume_B(i.e., the sample S to be measured)_Measuring)、S_{Yin (kidney)}He Yang S_{Yang (Yang)}Separately loading a detection tube T_Measuring、T_{Yin (kidney)}And T_{Yang (Yang)}Then adding equivalent vibrio parahaemolyticus virulent phage suspensions respectively and mixing uniformly;

4) will detect the tube T_Measuring、T_{Yin (kidney)}And T_{Yang (Yang)}Simultaneously and respectively putting the three detection channels into the chemical and biological process on-line monitoring system, and respectively recording the initial capacitive coupling non-contact conductance response values sigma of the detected liquid in the three detection tubes_{Measure beginning}、σ_{Beginning of yin}And σ_{Yang beginning}(ii) a After standing for 30min, respectively recording the end-point capacitance coupling non-contact conductance response values sigma of the measured liquid in the three detection tubes_{Test terminal}、σ_{Yin Final}And σ_{Yang Final}。

5) Respectively calculating S according to the following formula_Measuring、S_{Yin (kidney)}And S_{Yang (Yang)}The capacitance coupling non-contact conductivity response change value:

Δσ_measuring＝σ_{Test terminal}-σ_{Measure beginning}

Δσ_{Yin (kidney)}＝σ_{Yin Final}-σ_{Beginning of yin}

Δσ_{Yang (Yang)}＝σ_{Yang Final}-σ_{Yang beginning}

6) Will delta sigma_MeasuringSubstituting the numerical value into a working curve function relation to obtain the quantity (concentration) of the vibrio parahaemolyticus in the water body to be detected; according to σ_{Yin (kidney)}Less than or equal to 2 mu S/cm and delta sigma_{Yang (Yang)}The judgment experiment of more than or equal to 10 mu S/cm has no false positive and false negative.

Further, the working curve is formulated as follows:

1) according to the preparation of a positive control sample S_{Yang (Yang)}Respectively prepared by the same method as described above, respectively containing 10¹⁰、10⁹、10⁸、10⁷、10⁶、10⁵And 10⁴Bacterial suspension S of CFU/ml vibrio parahaemolyticus_{Yang 10}、S_{Yang 9}、S_{Yang 8}、S_{Yang 7}、S_{Yang 6}、S_{Yang 5}And S_{Yang 4}；

2) Take the same volume S_{Yang 10}、S_{Yang 9}、S_{Yang 8}、S_{Yang 7}、S_{Yang 6}、S_{Yang 5}、S_{Yang 4}And negative control S_{Yin (kidney)}Separately loaded into the detecting tubes T_{Yang 10}、T_{Yang 9}、T_{Yang 8}、T_{Yang 7}、T_{Yang 6}、T_{Yang 5}、T_{Yang 4}And T_{Yin (kidney)}Then adding suspensions of equivalent vibrio parahaemolyticus virulent phage VB _ VpS _ PG08 respectively, and mixing uniformly;

3) will detect the tube T_{Yang 10}、T_{Yang 9}、T_{Yang 8}、T_{Yang 7}、T_{Yang 6}、T_{Yang 5}、T_{Yang 4}And T_{Yin (kidney)}Simultaneously and respectively inserting 8 detection tubes into 8 detection channels of the chemical and biological process on-line monitoring system, and respectively recording initial capacitive coupling non-contact conductance response values sigma of the detected liquid_{Yang 10 Yuan}、σ_{Yang 9 Yuan}、σ_{Yang 8 Yuan}、σ_{Yang 7 Yuan}、σ_{Yang 6 initial stage}、σ_{Yang 5 Yuan}、σ_{Yang 4 initial stage}And σ_{Beginning of yin}(ii) a After standing for 30min, respectively recording the end point capacitance coupling non-contact conductance response values sigma of the detected liquid in 8 detection tubes_{Yang 10 Final}、σ_{Yang 9 Final}、σ_{Yang 8 Final}、σ_{Yang 7 Final}、σ_{Yang 6 Final}、σ_{Yang 5 Final}、σ_{Yang 4 Final}And σ_{Yin Final}；

4) According to the formula delta sigma-sigma_{Final (a Chinese character of 'gan')}-σ_{First stage}Separately calculate S_{Yang 10}、S_{Yang 9}、S_{Yang 8}、S_{Yang 7}、S_{Yang 6}、S_{Yang 5}And S_{Yang 4}Initial response value sigma of capacitive coupling non-contact conductance_{First stage}And endpoint response value σ_{Final (a Chinese character of 'gan')}The difference between the two and plotting the data obtained against logC (concentration of Vibrio parahaemolyticus) to obtain a functional relationship between Δ σ and logC; at the same time, according to Δ σ_{Yin (kidney)}The test is judged to have no false positive result less than or equal to 2 mu S/cm.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention has higher automation degree, accurate result and good reproducibility, but only needs portable instruments and equipment to realize on-site measurement.

2. The method does not need the steps of separation, culture and the like, and can be used for measuring the qualitative and quantitative information of the vibrio parahaemolyticus in the culture water body in one step.

3. The invention has higher detection efficiency, only needs less than 2h (single sample) from the collection of the sample to the acquisition of the result, and can simultaneously detect up to tens of samples.

4. The invention has simple operation and easy learning, does not need special training and is easy to popularize.

Description of the drawings:

FIG. 1: the relationship between the conductivity response difference Δ σ and logC (concentration of Vibrio parahaemolyticus).

The specific implementation mode is as follows:

the technical process of the present invention will be described and explained in detail by the following examples, which are not intended to limit the present invention in any way.