Substance, kit and method used for detecting systemic infection
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A systemic infection and kit technology, applied in the material field of systemic infection detection, can solve problems such as high cost, low ratio, and insufficient sequencing depth, and achieve high detection sensitivity and specificity, and good severity effects
Active Publication Date: 2017-11-03
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 source of the sample is a middle-aged male patient, who was clinically diagnosed with laryngeal cancer surgery, severe pneumonia, and septic shock. Sputum and blood cultures showed Escherichia coli.
[0042] 1. Collect peripheral blood and centrifuge to separate plasma
[0043] Use cfDNA-specific blood collection tubes to routinely collect 6ml of peripheral blood and store it as required; centrifuge at 2500g for 10 minutes, then extract the supernatant plasma, centrifuge at 20000g for 10 minutes, and extract 3ml of supernatant plasma.
[0044] 2. Use the kit QIAamp Circulating Nucleic Acid Kit to extract plasma circulating DNA as required
[0045]1. Add 3ml plasma, 300μl Proteinase K, and 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 extr...
Embodiment 2
[0119] The source of the sample was an elderly male patient who was clinically diagnosed with 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] Use cfDNA-specific blood collection tubes to routinely collect 6ml of peripheral blood and store it as required; centrifuge at 2500g for 10 minutes, then extract the supernatant plasma, centrifuge at 20000g for 10 minutes, and extract 3ml of supernatant plasma.
[0122] 2. Use the kit QIAamp Circulating Nucleic Acid Kit to extract plasma circulating DNA as required
[0123] 1. Add 3ml plasma, 300μl Proteinase K, and 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 Q...
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Abstract
The invention relates to a substance, a kit and a method used for detecting systemic infection. The substance is a circulating DNA disintegrated and released in vivo from a pathogenic bacterium. The substance provided by the invention is based on the detection of DNA degraded by the pathogenic bacterium of peripheral blood, objectively reflects that whether the pathogenic bacterium invades into blood circulation, and better helps to distinguish the systemic infection and non-infective diseases. The substance does not depend on the separation and identification of a living bacterium thallus, is not affected by the content of the living bacterium in a blood specimen, is not affected by the influences of the contaminated bacterium in specimen collection process, and is higher in detection sensitivity and specificity. The provided detection kit comprises a capture probe for 16 S rDNA of 662 pathogenic bacteria, and can better identify the species of the pathogenic bacterium. The provided detection method better reflects the severity of the infection as well as the course and the condition of the disease through the semi-quantitative analysis of the circulating DNA from the bacterium in the peripheral blood, so as to guide treatment and evaluate 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 complication with high morbidity, high mortality, and high treatment costs. A clear etiological diagnosis plays a vital role in guiding anti-infective treatment and improving the prognosis of patients. At present, clinically, etiological diagnosis mainly relies on the isolation and culture 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 the sensitivity is too low, and the positive rate is less than 30%. In addition, due to the contamination factors during the specimen collection process, this detection method still has a certain false positive rate. [0003] The positive detection rate of blood culture is mainly affected by th...
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