Primer group, kit and library construction method for detecting five bloodstream infection pathogens,

Abstract

The invention discloses a primer group, a kit and a library construction method for detecting five bloodstream infection pathogens. The primer group comprises primer pairs used for detecting staphylococcus aureus, escherichia coli, acinetobacter baumannii, klebsiella pneumonia and enterococcus faecium. The primer group disclosed by the invention is high in specificity, a situation of non-specificamplification is effectively reduced, and the culture level of bacteria can be directly authenticated through specific primer amplification. When the primer group disclosed by the invention is adoptedto detect the bloodstream infection pathogens, a blood culture operation is not required, pathogens in a blood sample can be directly detected, the shortest time for obtaining a detection result is 48 h, and a detection period is greatly shortened.

Description

technical field [0001] The invention relates to the technical field of pathogenic microorganism detection, in particular to a primer set, a kit and a method for building a library for detecting five bloodstream infection pathogens. Background technique [0002] Bloodstream infection is a serious systemic infectious disease. The current clinical diagnosis method is still based on blood culture, which takes a long time and has a low positive culture rate, which is not conducive to timely diagnosis and medication judgment. Clinically, drugs are often prescribed based on doctors’ empirical diagnosis. On the one hand, it increases the economic burden of patients, and on the other hand, it also increases the risk of drug resistance caused by antibiotic abuse. Therefore, accurate and rapid detection of pathogens is the current trend of clinical diagnosis. [0003] Using the method of multiple target enrichment can realize the simultaneous detection of different targets in the sampl...

AI Technical Summary

Problems solved by technology

However, the identification of this method depends on the change of temperature, so the temperature sensitivity of the equipment is very high; in addition, only one sample can be detected at a time, and the throughput is low; the species judgment depends on the temperature change, so the scalability of the identification of species Low

2) Prove-it in Finland TM Sepsis (Mobidiag, Finland) bloodstream infection detection product can identify 19 kinds of Gram-negative bacteria, 15 kinds of Gram-positive bacteria, 8 kinds of fungi and drug-resistant genes within 3.5 hours through multiplex PCR and hybridization. Multiplex PCR itself has technical limitations and low scalability. In addition, hybridization-based methods are prone to non-specific hybridization and affect the accuracy of detection results.

However, due to the limitations of 16S identification, some targets could not be identified to the species level

4) Luminex Bloodstream Infection Pathogen Multiple Detection Kit ( Liquid phase chip method) is a method based on multiplex PCR and liquid phase capture to realize the detection of specific pathogens, but due to the limitation of fluorescence detection resolution of this method, the detection sensitivity is not high

[0005] The existing bloodstream infection detection technology or products have the following defects: 1) The scalability of detecting pathogens is low

The multiplex PCR detection technology represented by Filmarray, after multiplex PCR amplification, can be distinguished by melting curve or detected by fluorescent signal. Due to the limitations of the technology itself, the expansion of the multiplex PCR system may affect the system on the one hand. On the other hand, it will increase the load of subsequent signal detection, so this type of technology has poor scalability and high technical limitations

2) Pathogen resolution is low

MagicPlex by Seegene TM Take sepsis real-time detection as an example. Since 16S primers are used for detection, some primers can only identify species at the genus level, but cannot be accurate to the species level, and cannot accurately distinguish specific species.

3) The false positive rate of test results is high

Most of the molecular multiple detection products currently on the market detect target molecules through indirect detection of sequence signals, during which false positives may occur due to non-specific amplification or other chimerism

4) Low detection throughput

Taking Filmarray as an example, a single test can only detect one sample, which is far from meeting the clinical throughput requirements

5) Low detection sensitivity

Filmarray detection sample type is blood culture, which is suitable for detection of high-concentration target samples; Luminex bloodstream infection pathogen multiplex detection kit can directly detect whole blood samples, but its detection sensitivity is low, generally only up to 10^ 4 to 10^5 copies / ml

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