Primer and probe combination and kit for detecting human pathogenic bacteria
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A technology of pathogenic bacteria and kits, applied in the field of medical detection, can solve the problem that the detection sensitivity cannot reach the clinical level
Pending Publication Date: 2019-12-13
PILOT GENE TECH HANGZHOU CO LTD
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Embodiment 1
[0058] Embodiment 1 Primer and probe combination of the present invention, reference primer and probe combination
[0059] The sequences of primers and probes are listed in Table 1.
[0060] Table 1 Primer and Probe Combination Sequences
[0061]
[0062]
Embodiment 2
[0063] Embodiment 2 Kit of the present invention
[0064] The kit includes primers and probe combinations of the present invention in Table 1, sample extraction reagents, multiple digital PCR detection reagents, positive control substances, negative control substances, PCR chips, special oil for chips, ddH 2 O.
[0065] Among them, the negative control substance is ultrapure water, and the positive control substance is the standard substance corresponding to the target strain detected by the kit.
Embodiment 3
[0067] Utilize the kit of embodiment 2 to Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecium, Staphylococcus hominis and Baumannii in 84 cases of blood samples Acinetobacter was tested. Among them, 14 were positive samples and 70 were negative samples. At the same time, the blood culture method is used to determine the infection of the sample, which is the current industry gold standard.
[0068] The detection steps are:
[0069] 1. Nucleic acid extraction:
[0070] The instruments and equipment used in the nucleic acid extraction process are shown in Table 2, and the reagents are shown in Table 3.
[0071] Table 2 Instrument and Equipment List
[0072]
[0073] Table 3 Reagent name and storage
[0074] Reagent name storage Lysate A Store at 2-8°C Proteinase K Store at 2-8°C magnetic bead suspension Store at 2-8°C Washing solution A Store at 2-8...
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Abstract
The invention relates to the technical field of medical detection, and in particular, relates to a primer and probe combination and a kit for detecting human pathogenic bacteria. The kit comprises theprimer and probe combination for detecting pseudomonas aeruginosa, escherichia coli, klebsiella pneumoniae, staphylococcus epidermidis, staphylococcus aureus, enterococcus faecium, human staphylococcus and acinetobacter baumannii. According to the kit, the primer and probe combination is adopted, multiple digital PCR is combined, the pathogenic bacteria types and the target nucleic acid copy number in the detection range can be rapidly and accurately identified, the detection method has high sensitivity, and positive detection of low-copy nucleic acid fragments of pathogenic bacteria is guaranteed; meanwhile, the variety of the pathogenic bacteria or the change of the copy number of the target nucleic acid fragment in the body of a pathogenic bacteria infected patient can be dynamically monitored, the treatment effect is assisted and evaluated in time, and reference is provided for optimization of a doctor clinical scheme.
Description
technical field [0001] The invention relates to the technical field of medical detection, in particular to a combination of primers and probes and a kit for detecting human pathogenic bacteria. Background technique [0002] Human pathogenic bacteria include bacteria, viruses, spirochetes, rickettsia, chlamydia, mycoplasma, fungi and actinomycetes, etc. Generally speaking, pathogenic bacteria refer to bacteria in pathogenic microorganisms. While the vast majority of bacteria are harmless or even beneficial, a significant number can cause disease. When the human body's immunity is low or there are wounds, pathogenic bacteria can invade tissue parts, leading to blood, urine and cerebrospinal fluid infections. For example, Staphylococcus aureus and Streptococcus are also normal flora, and can often exist on the skin and nasal cavity without causing disease. However, under certain conditions, clinical patients may suffer from serious infectious diseases such as pneumonia, mening...
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