Construction method of DNA library for detecting pathogens in respiratory tract sample and kit

Abstract

The invention belongs to the technical field of biology, and relates to a construction method of a DNA library for detecting pathogens in a respiratory tract sample and a kit, and the construction method comprises the following steps: carrying out fragmentation treatment on a DNA sample and an internal label corresponding to the DNA sample through a one-step reaction, so as to realize terminal repair, phosphorylation and A addition on the DNA sample and the internal label, and obtaining a first product; sequentially carrying out joint connection and first purification treatment on the first product to obtain a second product; sequentially carrying out PCR enrichment and secondary purification treatment on the second product to obtain the DNA library for detecting the pathogens in the lower respiratory tract sample. The DNA library construction process is simple, convenient and rapid, and the method can be suitable for DNA library construction with low initial quantity and poor quality; and an interior label is added so as to facilitate the interpretation of a subsequent raw signal analysis result.

Description

technical field [0001] The application relates to the field of biotechnology, in particular to a method and a kit for constructing a DNA library of pathogens in respiratory samples. Background technique [0002] There are significant differences in the proportion of nosocomial infections among departments in Chinese hospitals. Some studies have pointed out that the most susceptible departments for nosocomial infections are surgical ICU, hematology department, emergency department, respiratory medicine department, and neurology department. The age distribution of infected patients is V-shaped, mainly infants and young children under two years old and elderly patients over 60 years old. The immune systems of infants and young children are immature, while elderly patients are critically ill, have relatively low immune function, have more invasive procedures, and often have underlying diseases. Therefore, these two groups are currently high-risk groups for nosocomial infection. ...

AI Technical Summary

Problems solved by technology

The above steps include three steps of purification. The library building step takes a long time. Multiple magnetic bead purifications will inevitably increase the loss of DNA samples, so the requirements for the total amount and quality of DNA are high.

[0005] At present, the kits for fragmentation, end repair and one-step reaction of adding A in the market all amplify the amount of template to varying degrees and cannot truly reflect the composition of nucleic acids in the sample.

[0006] The host ratio of lower respiratory tract samples such as alveolar lavage fluid and sputum varies greatly, and there are a large number of colonized bacteria. The existing reagents lack internal standards to normalize the results, which will affect the accuracy of later data analysis.

[0007] It can be seen that the existing library construction steps are cumbersome, time-consuming for multiple magnetic bead purification, high requirements on the total amount and quality of DNA, fragmentation, end repair and adding A steps cannot be integrated, or can be integrated There is also a certain degree of template volume amplification

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