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Processing method for sample for detecting bloodstream infection pathogenic bacteria

A treatment method and technology for pathogenic bacteria, applied in the field of clinical detection, can solve the problems of detection methodology failing to achieve positive detection and low detection sensitivity, and achieve the effect of ensuring the positive detection rate

Pending Publication Date: 2019-09-03
PILOT GENE TECH HANGZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its detection sensitivity is low. For patients with clinical bloodstream infection, the content of pathogenic bacteria in their blood samples is not more than 10CFU / mL, or even lower, and its detection methodology often cannot achieve the effect of positive detection

Method used

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  • Processing method for sample for detecting bloodstream infection pathogenic bacteria
  • Processing method for sample for detecting bloodstream infection pathogenic bacteria
  • Processing method for sample for detecting bloodstream infection pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: Sample processing method of the present invention

[0034] 1. Bacteria isolation

[0035] Carry out bacterial cell separation according to the reagent in Table 1;

[0036] Table 1

[0037]

[0038]

[0039] Use red blood cell lysate to lyse the patient's plasma sample, the ratio of blood sample to red blood cell lysate is 1:3, incubate at 4 degrees for about 10 minutes, and centrifuge the lysed sample to obtain the precipitate containing bacteria and white blood cells;

[0040] The precipitate was added to the blood cell lysate at a ratio of 1:1, 20-50 microliters of fluorinated oil was added, and the mixture was fully shaken and mixed.

[0041] Add proteinase K to cleavage the protein, incubate at 55°C for 5-10 minutes, centrifuge at 12000rpm for 10 minutes, and separate the bacteria.

[0042] The centrifuged sample was carefully removed to obtain the fluorinated oil and the precipitate of the lower layer to obtain an isolated sample.

[0043] 2...

Embodiment 2

[0052] Example 2: Comparison between the sample processing method of the present invention and the conventional blood culture method

[0053] Escherichia coli artificially mixed with 5-10 CFU into the blood as a simulated sample, respectively processed according to the sample processing method of the present invention (Example 1) and the conventional blood culture method, and then amplified under the same droplet digital PCR conditions , the results are shown in Table 2;

[0054] Table 2

[0055]

[0056] The above experimental results show that under the same trace bacterial concentration conditions, the samples obtained by the sample processing method of the present invention can obtain more positive points after testing, while the conventional blood culture method cannot obtain positive points, indicating that the method of the present invention can obtain more positive points. Ensure the positive detection rate of trace pathogenic bacteria.

Embodiment 3

[0057] Embodiment 3: Comparison of different blood cell lysates

[0058] Escherichia coli (9CFU) mixed in the blood is still used as a simulated sample in the past, and the simulated sample is divided into three parts, and processed with reference to the method of Example 1. The difference is that the blood cell lysate is different, and then under the same droplet digital PCR conditions Amplification, the results are shown in Table 3 and Figure 1-3 ;

[0059] table 3

[0060] group blood cell lysate Number of positive amplification points 1 2.74% sorbitol in RNase-free aqueous solution 61 2 5M guanidine hydrochloride in RNase-free aqueous solution 73 3 5% SDS in RNase-free aqueous solution 34

[0061] Depend on Figure 1-3 As can be seen from the results in Table 3, under the premise of the same trace concentration of pathogenic bacteria, more positive amplification points can be obtained by using guanidine hydrochloride and sorbit...

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Abstract

The invention relates to the technical field of clinical detection, and discloses a processing method for a sample for detecting bloodstream infection pathogenic bacteria. The method comprises the steps: lysing erythrocytes of a to-be-tested sample, then adding a hemocyte lysate and an indication solvent into an obtained precipitate, mixing evenly, lysing proteins and incubating, centrifuging to obtain the indication solvent and an underlying precipitate, and thus obtaining a separated sample; and simultaneously extracting RNA and DNA from the separated sample, and finally obtaining a mixed nucleic acid solution. The erythrocytes are lysed firstly, and then lysis is performed with the hemocyte lysate, so pathogenic bacteria can be guaranteed to be not lysed to the greatest degree, and theseparation of the pathogenic bacteria in the blood sample is efficiently guaranteed; with use of the characteristic that the level of RNA in living bacteria is much higher than the copy number of genomic DNA, the genomic DNA and RNA of the pathogenic bacteria are extracted simultaneously. Compared with sample pretreatment in a conventional pathogenic bacteria detection scheme, the positive detection rate of trace pathogenic bacteria can be guaranteed.

Description

technical field [0001] The invention relates to the technical field of clinical detection, in particular to a sample processing method for detecting bloodstream infection pathogenic bacteria. Background technique [0002] Bloodstream infection (BSI) is one of the clinically serious infectious diseases. According to different symptoms, it can be divided into sepsis, sepsis and septic shock. It has a high morbidity and mortality rate. The common sources of infection include bacteria and fungi. At present, the gold standard for the diagnosis of bloodstream infection is blood culture, which identifies pathogens by biochemical methods or microscope observation, but this method is time-consuming, usually taking several days to confirm the results, and for pathogens that are difficult or impossible to culture, it is difficult to Accurate detection. In the absence of accurate diagnostic results, clinicians may blindly use antibiotics in order to alleviate symptoms, resulting in re...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/04
CPCC12Q1/6806C12Q2521/107C12Q2521/537
Inventor 朱留伟夏江董德坤
Owner PILOT GENE TECH HANGZHOU CO LTD
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