Processing method of fluorescence intensity data in fluorescence droplet detection

Abstract

The invention discloses a processing method of fluorescence intensity data in fluorescence droplet detection. The method is used for the nucleic acid detection based on fluorescence droplet. The method comprises the steps of step 1, acquiring the fluorescence intensity data of all fluorescence droplets, and preprocessing the data, step 2, conducting a primary classification of the data, step 3, based on the result of the primary classification, acquiring the fluorescent intensity distribution of the negative droplets and the positive droplets, step 4, calculating final decision threshold value t, step 5, using the decision threshold value t for a second time classification of data, step 6, calculating a sample concentration. The processing method accurately describes the distribution of the fluorescent intensity of digital PCR fluorescent micro-drop, and can automatically determine the suitable threshold value automatically to identify the quantity of the positive micro-droplets without the need of negative control. The processing method can effectively enhance the accuracy of data classification, thus considerably enhancing the accuracy of the fluorescence droplet detection result.

Description

technical field [0001] The invention relates to the technical field of processing methods for fluorescence intensity data in fluorescent droplet detection, in particular to an automatic positive droplet identification method based on fluorescent droplet nucleic acid detection. Background technique [0002] Polymerase Chain Reaction (PCR) is an in vitro nucleic acid amplification technique developed in the mid-1980s. It has the characteristics of strong specificity, high sensitivity, easy operation, and time saving. It can be used in basic research such as gene isolation, cloning and nucleic acid sequence analysis. Digital PCR is a new generation of quantitative PCR analysis technology that has developed rapidly in recent years. It uses microfluidic technology to disperse a large number of diluted nucleic acid solutions into microreactors on the chip. The number of nucleic acid templates in each reactor is less than or equal to 1 . In this way, after the PCR cycle, the reac...

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Problems solved by technology

Therefore, accurately determining the fluorescence threshold has an important impact on the final result. At present, there are two methods for determining the threshold based on fluorescent microdroplet PCR nucleic acid detection. One is to use a negative quality control without the sample to be tested. The maximum fluorescence intensity of the microdroplets determines the discrimination threshold. This judgment method has high recognition accuracy, but it needs to refer to the negative quality control; the other is based on the Kmeans clustering algorithm. This method does not require comparison, but because the distribution of fluorescent microdroplets is not Simple two types, but there are many kinds of interference, the final result often has a large deviation

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