Multiplex fluorescence PCR kit, method and application for detecting respiratory tract pathogens
A multiple fluorescence and kit technology, applied in the field of biomedicine, can solve the problems of narrow detection range of respiratory virus nucleic acid detection kits, limited detection throughput by instruments, limited use range, etc., so as to shorten the detection time and reduce the detection cost. , high sensitivity and specificity
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[0050] Example 1
[0051] This example provides a multiplex real-time PCR detection kit for detecting adenovirus, cytomegalovirus, boca virus and Mycoplasma pneumoniae, including a probe for detecting adenovirus and a specific amplification of adenovirus target gene A primer pair for detecting cytomegalovirus, a probe for detecting cytomegalovirus and a primer pair for specifically amplifying a target gene of cytomegalovirus, a probe for detecting Boca virus and a primer pair for specifically amplifying a target gene of Boca virus, Probe for detection of Mycoplasma pneumoniae and primer pair for specific amplification of Mycoplasma pneumoniae target gene, MgCl 2 , dNTPs Mixture, Taq DNA polymerase, plasmid mixture containing target gene and nuclease-free water;
[0052] The present embodiment provides a method for detecting bronchial lavage fluid using the above-mentioned kit:
[0053] 1. Extract the nucleic acid from the bronchial lavage fluid of the clinical sample, and ob...
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[0069] Example 2
[0070] This example is the same as Example 1, except that the clinical samples, the primer and probe concentrations for amplification of each pathogenic microorganism, the system component concentrations of the amplification reaction, and the conditions of the amplification reaction are different from those in Example 1: this implementation The clinical sample used is sputum, the primer concentration for each pathogenic microorganism amplification is 0.1 μM, and the probe concentration is 50 nM; MgCl in the reaction buffer described in the amplification reaction system 2 The working concentration of dNTPs Mixture is 2mM, the working concentration of dNTPs Mixture is 350μM, the working concentration of TaqDNA polymerase is 4U / μL, and the amplification conditions are shown in the following table:
[0071]
[0072] The CT value of cytomegalovirus in this example is ≤40, the detection results of the negative control, adenovirus, boca virus and Mycoplasma pneu...
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[0073] Example 3
[0074] This example is the same as Example 1, except that the clinical samples, the primer and probe concentrations for amplification of each pathogenic microorganism, the component concentrations in the amplification reaction system, and the conditions of the amplification reaction are different from those in Example 1: this implementation The clinical sample used in this example is bronchoalveolar lavage fluid, the primer concentration for each pathogenic microorganism amplification is 1.0 μM, and the probe concentration is 250 nM; MgCl in the reaction buffer 2 The working concentration of dNTPs Mixture is 4mM, the working concentration of dNTPs Mixture is 450μM, and the working concentration of Taq DNA polymerase is 6U / μL; the amplification conditions are shown in the following table:
[0075]
[0076] The CT value of Bocavirus in this example is ≤40, the detection results of the negative control, adenovirus, Bocavirus and Mycoplasma pneumoniae have no...
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