Pathogenic microorganism detection primer set, kit and method based on targeted sequencing

Abstract

The invention discloses a pathogenic microorganism detection primer set based on targeted sequencing, belonging to the technical field of microorganism detection. The pathogenic microorganism detection primer set includes the 1st primer pair to the 56th primer pair, wherein, the nth primer pair is composed of the nth forward amplification primer shown in SEQ ID No. (2n-1) and SEQ ID No.2n The composition of the nth reverse amplification primer shown, n=1-56. The invention also discloses a pathogenic microorganism detection kit comprising the primer set, and a multiple PCR amplification method for detecting pathogenic microorganisms by using the primer set or the kit. Using the invention to detect pathogenic microorganisms has the advantages of comprehensive coverage of pathogens, high sensitivity, high detection throughput, and low cost, and can assist in clinical and accurate identification of common pathogens, covering bacteria, fungi, DNA viruses, RNA viruses, parasites, mycoplasma chlamydia, etc. , has important clinical application value.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, and in particular relates to a primer set, a kit and a method for detection of pathogenic microorganisms based on targeted sequencing. Background technique [0002] At present, the diagnosis level of infectious diseases in my country needs to be improved urgently. Statistics in 2017 show that the incidence and mortality of infectious diseases in my country are 509.54 (1 / 100,000) and 1.43 (1 / 100,000) respectively, and they still maintain an upward trend. Compared with the world, the level of diagnosis and treatment of infectious diseases in my country is relatively low. The prevalence and death rates of HIV and TB are 3.6 (1 / 100,000) and 0.76 (1 / 100,000), respectively, which is far behind developed countries. Among the patients with infectious diseases in my country every year, more than 300 million patients need to use pathogen detection methods to check for further auxiliary diag...

AI Technical Summary

Problems solved by technology

These technologies have played an important role in the daily identification of pathogenic microorganisms, but there are also certain shortcomings: the former needs to rely on cultivation, the cycle is long, and the identification accuracy is low; the latter requires certain prior knowledge of microbial sequences and cannot achieve high-throughput detection, etc.

Compared with traditional culture methods, mNGS has higher sensitivity and faster detection speed, but in practical applications, there are still some difficult problems, which also limit its wide application:

[0005] 1. It is impossible to accurately distinguish whether the detected microorganisms are colonizing bacteria, background bacteria, or pathogenic bacteria;

[0006] 2. mNGS technology is less sensitive to the detection of intracellular bacteria and fungi: Due to the limitation of sequencing costs, mNGS needs to eliminate the interference of human cells as much as possible

Therefore, for intracellular infectious bacteria such as Mycobacterium tuberculosis and Legionella, the detection sensitivity is low due to their low density in body fluids; at the same time, mNGS has low extraction efficiency for pathogens and fungi with thicker cell walls. lead to lower detection rate and sensitivity

[0007] 3. It is difficult to detect RNA viruses: RNA transcription itself has higher abundance and complexity, is easy to degrade, and has high requirements for transportation and storage. Therefore, there are still certain difficulties in the clinical detection of RNA viruses

[0008] 4. Standards are difficult to unify: In terms of the reliability of sequencing results, different sequencing companies use different reporting methods for reliable data, such as depth, coverage, abundance, estimated concentration, and confidence. no uniform standard

[0009] 5. High host background: For general sample types such as blood, alveolar lavage fluid, sputum, ascites, etc., the host genome contamination ratio is generally above 80%, which results in very few microbial sequences detected, and a large part of the data are cannot be used effectively

[0010] 6. High cost: At present, mNGS single detection (DNA or RNA), terminal charges are relatively expensive, such as non-dangerous and critically ill patients, it is generally difficult to receive detection

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