Primer pool, kit and method for detecting bloodstream infection by targeting sequencing method

Abstract

The invention discloses a primer pool, a kit and a method for detecting bloodstream infection by targeting high-throughput sequencing based on a multiple amplification method. The primer pool comprises a forward single-molecule tag primer and a reverse single-molecule tag primer, and each of the primers structurally comprises six parts, namely a forward single-molecule tag primer and a reverse single-molecule tag primer, and the forward single-molecule tag primer and the reverse single-molecule tag primer respectively comprises six parts: a tag universal amplification primer, a molecular tag UMI, a target specific primer sequence, an RNA residue, a DNA protection base and a blocking group sequentially arranged from a 5 '-3' end. The kit comprises the primer pool and further comprises a unique enzyme preparation and a reaction buffer. The kit can perform absolute quantification, has high specificity, can significantly reduce primer dimers, can increase or decrease primers at any time according to expected target requirements, has strong expansibility, can simultaneously perform target co-detection of RNA and DNA in a single tube, has comprehensive coverage, and reduces the detection cost.

Description

Technical field [0001] The present invention relates to the field of clinical detection techniques of infective diseases, and in particular, to a primer pool, kit and method for detecting blood flow induced high-throughput sequencing based on multi-expanded method. Background technique [0002] Hematic infection is a serious infectious disease. In my country's blood flow infection bacteria constitute 50%, Gram-negative bacteria accounted for 49.8%, anaerobic bacteria accounted for 0.2%. The microbiological identification of fungal blood is relatively difficult, and the total mortality rate of ICU Candidthiopathylin is 30-50%. White Candida, Tropical Candida, Light Slim Pearhaloni, Kuji Alpinee, Novel Intrackroccus occupied 95 to 98% of yeast fungal blood disease. Sepsis is also called sepsis, which is caused by host reaction caused by pathogens, which in turn leads to symptoms of circulatory dysfunction and organ function damage. Early sepsis can not get timely and effective cont...

AI Technical Summary

Problems solved by technology

In recent years, there have also been reports on the use of multiplex PCR library construction kits for the detection of pathogenic microorganisms, but most of them only detect part of the pathogens in a certain type of sample

In addition, because there are hundreds or even thousands of primers in the same reaction tube in the existing pathogenic microorganism multiple PCR method library construction kit, and the primer set and reaction system of the whole kit are the results of overall analysis optimization, so However, the existing pathogenic microorganism multiplex PCR library construction kits still have the following common problems: (1) primers interfere with each other, forming a large number of dimers, which reduces the performance of the kit; (2) non-specific amplification, resulting in a large number of non-target Amplicons affect subsequent sequencing; (3) accurate quantification cannot be performed; (4) amplification uniformity cannot be guaranteed; (5) poor scalability and flexibility

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