Method for determining the abundance of highly active heparin with respect to porcine small intestinal mucosa heparin IIa

By employing techniques such as the PowerSoil reagent kit, PCR amplification, Illumina NovaSeq 6000 sequencing, and the LEfSe algorithm, the problems of long detection cycles and inaccurate results in the detection of highly active heparin against porcine small intestinal mucosa in existing technologies have been solved. This has enabled high-sensitivity and low-cost heparin abundance determination, improving the quality of heparin products and controlling procurement costs.

CN122382183APending Publication Date: 2026-07-14HUANGCHUAN PENGSHENG ANIMAL PROD CO LTD
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Patent Information

Authority / Receiving Office
CN Β· China
Patent Type
Applications(China)
Current Assignee / Owner
HUANGCHUAN PENGSHENG ANIMAL PROD CO LTD
Filing Date
2026-02-24
Publication Date
2026-07-14

AI Technical Summary

Technical Problem

Existing technologies for detecting highly active heparin against IIa in porcine small intestinal mucosa suffer from problems such as long experimental cycles, large error in results, inaccurate detection results, and complex processes, leading to fluctuations in heparin product quality and high procurement costs.

Method used

Porcine small intestinal mucosal cells were lysed using the PowerSoil kit, followed by PCR amplification, high-throughput sequencing using an Illumina NovaSeq 6000 sequencer, noise reduction using the DADA2 workflow, annotation from the Greengenes database, and screening for differentially expressed microbiota using the LEfSe algorithm. A random forest model was then used to predict heparin activity, achieving high sensitivity and accuracy in detection.

Benefits of technology

This method enables the determination of high-anti-IIa heparin abundance in porcine small intestinal mucosa with short detection time, low cost, and accurate results. It is suitable for multi-point sampling studies and improves the stability of heparin product quality and control of procurement costs.

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Abstract

The application belongs to the technical field of heparin extraction, and particularly relates to a method for determining the abundance of high anti-IIa activity heparin of pig small intestinal mucosa. The method comprises the following steps: (1) crushing fresh pig small intestinal mucosa; (2) using a PowerSoil kit to lyse the mucosa cells crushed in step (1), and performing water bath at 50-65 DEG C for 10-20 min, and then adding an elution buffer to elute the DNA; (3) using PCR to amplify the eluted DNA solution; (4) using a Nextera XT DNA library preparation kit to construct a library, and then using an Illumina NovaSeq 6000 sequencer to perform high-throughput sequencing; (5) using a DADA2 process to denoise and remove chimeric sequences, and using a 97% similarity threshold to cluster to generate OTU, and using a Greengenes database to annotate; (6) then using an LEfSe algorithm to screen differential flora, and using a random forest model to predict heparin activity.
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