Research On The Key Technology Based On The Single Particle Detection Of Raw Milk SCC Test

ObjectiveThe somatic cells in raw milk are the cells of the dairy cows penetrate from the blood through the acinus.Under normal circumstances,the somatic cells counts(SCC)in raw milk is between 200,000 / ml and 300,000 / ml,about 98% of which are white blood cells.SCC below than 500,000 / ml is safety for mastitis nternationally,beyond that,the possibility of dairy cow suffering from mastitis increases.SCC as an important quality index in raw milk,on the one hand can help to monitor the health status of dairy cows breasts,on the other hand,is an effective guarantee for economic and security,because high SCC has a greater impact on the composition and quality of milk will.At present,somatic cells detection methods have direct and indirect two categories.Traditional indirect cell testing technology is the use of the physical and chemical properties of somatic cells to test,which has the advantage of economical,simple but generally with low detection accuracy.Direct microscopy is a gold standard for somatic cell testing,but it is cumbersome and time consuming.The cytometer based on the principle of single particle detection,with high degree of automation,speed and accuracy.However,due to the special liquid background of raw milk,and involving sample processing,the modules design of optical path and liquid line system,the somatic cells detection method based on single particle detection principle has not yet fully developed mature.In this paper,the fluorescence characteristics of raw milk samples were analyzed,and the optimal design method of optical path and liquid path modules which are mainly involved in single particle detection was discussed.The system performance index and counting accuracy of the prototype were tested and evaluated.Based on the principle of single particle detection,the standard process of somatic cell detection is put forwarded,and the key technology breakthrough on somatic cell detection platform is realized,which provides support for rapid and quantitative detection of somatic cells in raw milk.Research contents and methodsIn this paper,the somatic cells in raw milk were set as the target detection object,sample detection program was formulated after excluding the fluorescence of protein,fat globules and other particles which may exist in somatic cells disturbing detection.On the base of existing platform and technology of single-particle research group,this paper discusses the design of the key technology and the realization method of module unit of the single-particle detection system of somatic cells to optimize the design of the core module as the optical path and liquid line and realizes the somatic cell detection platform with engineering structures.The performance evaluation method of somatic cell detection single particle system is discussed and the relevant indexes are tested.Under the condition that the indexes are qualified,the experiment of counting accuracy is completed.Taking the traditional microscopic method as the comparation,the system can achieve accurate detection of somatic cell count in raw milk.Specific research content and research methods includes the following four aspects.1)Analysis and discussion of the fluorescence characteristics of the raw milk: singleparticle detection has a more stringent requirement for the background fluorescence of the sample solution to be analyzed.The autofluorescence signal, which is close to the fluorescence emission peak of the target,affects the detection result.Therefore,it is necessary to detect the autofluorescence of raw milk,and to analyze the endogenous fluorescence characteristics of potential interferences by fluorescence spectroscopy to find out the interference band that may affect the detection of single cell in somatic cells.The results of the analysis were used as the theoretical basis for the design of the sample detection program of somatic cell in the raw milk.The sample detection program including the system structure of the light source,the detection channel,fluorescent dye and the excitation wavelength was designed to ensure that the the somatic cells in raw milk can be accurately identified.2)Research on the key technology of optical path module in somatic cell single particle detection system: optical path module includes laser beam shaping and detection path two parts.In order to obtain a linear beam with a certain thickness and a gaussian distribution of energy,it is necessary to design the shaping optical channel and then simulate it based on the ZEMAX optical simulation software. According to the simulation results,the optical platform is designed to verify the experiment and obtain the energy distribution and size required detection spot.The optimal design of the single cell detection channel of the somatic cell includes the light collection and the confocal noise reduction.By analyzing the collected original signal data,the parameters of the detection channel are determined to improve the collection efficiency,signal-to-noise ratio and fluorescence sensitivity.3)Research on the key technology of liquid line module in somatic cell single particle detection system: It is mainly to optimize the flow cell and liquid piping system involved in single particle detection system.First,the flow velocity parameters of the sheath fluid were explored to ensure that the fluid flows in a stable laminar flow state in the flow cell with two-stage sheath fluid focusing design.Then,the sample fluid diameter under the different ratio of sheath fluid velocity to sample fluid velocity was observed by fluent simulation,than the optimum velocity ratio range was found,and the most stable flow section was found as the laser detection section. The optimization of the piping system is to analyze and improve the existing problems of the original pipeline module.At the end of each sample test,the automatic cleaning mechanism is added to realize the timely and effective two-way cleaning of the flow cell,pipe wall and syringe to reduce the milk sample liquid on the pipeline pollution,reduce the carry over rate and improve the reliability.4)Research on the performance evaluation method and relevant indexes test of somatic cell single particle detection system: somatic cell single particle detection system performance evaluation includs system indicators and counting accuracy. After the optimizing of optical path,the liquid circuit,the system modules are assembled together,and the single particle prototype system is tested according to the general requirements of the single particle technical indicators.It mainly includes the indexes of resolution,linearity,sensitivity and carrying over.The prototype system test is not only the validation of the optimization effect,but also the support and assurance for the reliability of somatic cell sample test.To verify the accuracy of the single-particle prototype system,the results were compared with the microscopic count method and the flow cytometry.Through the above four aspects research work,for the special inspection of raw milk somatic cells,the standard test process was put forwarded to achieve a special breakthrough in the platform for somatic cell testing,in order to realize a rapid and quantitative detection of somatic cells in raw milk.The innovation of this article.Firstly,on the basis of traditional hydraulic focus,two-stage hydraulic focusing design was proposed to improve the level of fluid confinement.Secondly,a single-laser single-channel thinning detection system based on the single particle detection principle was established,and a complete method for detecting somatic cells in raw milk was proposed for the first time in China.ResultThe results showed when the raw milk samples were excited at about 300 ~ 420 nm,the autofluorescence emission peak was about 525 nm.The fluorescence peak of somatic cells dyed with DAPI nucleic acid fluorescent dyes was about 460 nm excited by 405 nm lasers,which was well distinguished with the autofluorescence of raw milk.The results showed the detection of somatic cells was realized by using single fluorescent channels.The results show that the size of the laser spot in the detection area is about 100 × 20?m after beam shaping,and the energy density of the spot is Gaussian distribution.The simulation experiment is consistent with the experimental results obtained by the optical platform.The distance between the two cylinders and the detection area can be adjusted to obtain the Gaussian spot with different thickness range from 5?m to 30?m.The voltage response signal data of fluorescent microspheres(RCP-30-5A-2)at the PMT gain of 700 mV shows that,the S/N ratio of the detection channel with the confocal module increased by 0.57 times than without the confocal module,and the S/N ratio of the detection channel with the confocal module added the aperture,increased by 0.72 times compared to the the one without aperture.The results proved that detection channel with confocal and aperture has a better effect to the weak signal under large voltage gain.The results showed when the flow rate of the sample is 1 ?l/s and the ratio of sheath fluid / sample flow is in the range of 5 to 15 times,the sheath fluid and the sample solution are laminar flow under the focusing of the two-stage sheath fluid,and the better diameter of the sample within 10 ?m in theory.The results of FLUENT simulation show that compared with the same single-stage hydraulic focusing,the diameter of the sample flow after the two-stage hydraulic focusing is small obviously.The laminar flow in the middle of the test section is more stable and suitable for laser detection.The results showed that the CV value of the coefficient of variation increased with the increase of the sample flow velocity.The CV value was less than 2% when the sample flow velocity within 1 ?l/s.The linear correlation between the average fluorescence intensity of the particles in the fluorescence channel and the molecules of equivalent soluble fluorochrome carried by the fluorescent particles(RCP-30-5A)was R2 = 0.9921,and the fluorescence sensitivity of the CSB channel was 3.83 MESF.Three groups of repeated test results about carry over shows maximum C value was 0.80%,and the average value was 0.76%.The result of SCC accuracy experiment of raw milk samples show that centrifuge samples were generally lower than those without centrifugation,and the experimental results were significantly different(P<0.01).The SCC data difference between the single-particle prototype and flow cytometry(Thermo Fisher Scientific,Life,USA),fluorescence microscope method was not significant(P>0.05).The results of the singleparticle prototype were 91.92% of the fluorescence microscope method,98.64% of the flow cytometer.The standard deviation analysis of the repetitive experiments showed that the fluorescence microscope was more accurate than the flow cytometer and the single particle prototype when the concentration of somatic cells is low,however,the fluorescence microscope had the lowest accuracy and the single particle prototype had the highest accuracy when the concentration of somatic cells is high.ConclusionThe research results provide a feasible method for the rapid and convenient quantitative detection of somatic cells in raw milk,and realize the key technological breakthrough of the special platform for somatic cell detection.The performance evaluation method of somatic cell single particle detection system is put forward and the application field of single particle detection is extended,which provides a reference for the development of somatic cell detection technology based on single particle detection.