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A kind of substance, kit and method for systemic infection detection
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A technology of infection detection and kit, which is applied in the material field of systemic infection detection, can solve the problems of high cost, low ratio, and low clinical application feasibility, and achieve high detection sensitivity and specificity, and good severity Effect
Active Publication Date: 2022-05-03
SUN YAT SEN UNIV CANCER CENT
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Problems solved by technology
Recently, studies have reported that pathogen-derived circulating DNA was found in the peripheral blood of infected patients through whole-genome sequencing. However, due to insufficient sequencing depth, these studies did not elucidate the corresponding characteristics of pathogen-derived circulating DNA.
In our previous research, we found that the proportion of bacterial-derived circulating DNA in the peripheral blood of patients with systemic infection is extremely low, and the cost of direct whole-genome sequencing analysis is very high, and the clinical application is not feasible
However, other methods for the detection of circulating DNA of pathogen origin have not been reported so far
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Embodiment 1
[0041] The sample was from a middle-aged male patient with clinical diagnosis of postoperative laryngeal cancer, severe pneumonia, and septic shock. The results of sputum culture and blood culture were all Escherichia coli.
[0042] 1. Collect peripheral blood and centrifuge to separate plasma
[0043] Routinely collect 6ml of peripheral blood using a cfDNA special blood collection tube and store it as required; centrifuge at 2500g for 10 minutes, then extract the supernatant plasma at 20,000g for 10 minutes, and extract 3ml of supernatant plasma.
[0044] 2. Use the QIAamp Circulating Nucleic Acid Kit to extract plasma circulating DNA as required
[0045]1. Add 3ml plasma, 300μl Proteinase K, 2.4ml Buffer ACL (containing 1.0μg carrier RNA) to a 50ml centrifuge tube, vortex for 30s, and incubate at 60°C for 30min.
[0046] 2. Take out the centrifuge tube, add 5.4ml Buffer ACB, vortex for 30s, and incubate on ice for 5min.
[0047] 3. Connect the negative pressure extractor ...
Embodiment 2
[0119] The sample source was an elderly male patient with a clinical diagnosis of severe pneumonia. The sputum culture was Acinetobacter baumannii, and the blood culture result was negative.
[0120] 1. Collect peripheral blood and centrifuge to separate plasma
[0121] Routinely collect 6ml of peripheral blood using a cfDNA special blood collection tube and store it as required; centrifuge at 2500g for 10 minutes, then extract the supernatant plasma at 20000g for 10 minutes, and extract 3ml of supernatant plasma.
[0122] 2. Use the QIAamp Circulating Nucleic Acid Kit to extract plasma circulating DNA as required
[0123] 1. Add 3ml plasma, 300μl Proteinase K, 2.4ml Buffer ACL (containing 1.0μg carrier RNA) to a 50ml centrifuge tube, vortex for 30s, and incubate at 60°C for 30min.
[0124] 2. Take out the centrifuge tube, add 5.4ml Buffer ACB, vortex for 30s, and incubate on ice for 5min.
[0125] 3. Connect the negative pressure extractor QIAvac 24Plus, QIAamp Mini column ...
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Abstract
The invention relates to a substance, a kit and a detection method for systemic infection detection. The substance refers to the circulating DNA released from the disintegration of pathogenic bacteria in the body. The invention is based on the detection of degraded DNA of pathogenic bacteria in peripheral blood, objectively reflects whether there is pathogenic bacteria invading into blood circulation, and can better distinguish systemic infection and non-infectious disease. The invention does not rely on the separation and identification of viable bacteria, is not affected by the content of viable bacteria in blood samples, and is not affected by contaminated bacteria in the process of collecting samples, and has higher detection sensitivity and specificity. The detection kit provided by the present invention contains 662 kinds of capture probes of 16S rDNA of pathogenic bacteria, which can better identify the genus and species of pathogenic bacteria. The detection method provided by the present invention can better reflect the severity, course and outcome of the infection through the semi-quantitative analysis of the circulating DNA derived from bacteria in the peripheral blood, thereby guiding the treatment and evaluating the prognosis.
Description
technical field [0001] The present invention relates to a substance, kit and method for systemic infection detection. Background technique [0002] Systemic infection is a very common clinical comorbidity, with high morbidity, high mortality and high treatment costs. A clear etiological diagnosis plays a crucial role in guiding anti-infective treatment and improving patient prognosis. At present, in clinical practice, pathogenic diagnosis mainly relies on the isolation, culture and identification of pathogenic bacteria. Positive blood culture results are still the gold standard for the diagnosis of systemic infection. However, the biggest disadvantage of blood culture testing is that its sensitivity is too low, with a positive rate of less than 30%. In addition, due to the factors of contamination in the process of specimen collection, the detection method still has a certain false positive rate. [0003] The positive detection rate of blood culture is mainly affected by...
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