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Fluorescence quantitative PCR (polymerase chain reaction) detection method for 14 serotypes of Uu (ureaplasma urealyticum)

Ureaplasma urealyticum, fluorescence quantitative technology, applied in the field of genes, can solve the problems of cumbersome, difficult to widely develop, time-consuming, etc., to achieve the effect of convenient operation

Inactive Publication Date: 2014-08-06
ZHEJIANG UNIV
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Problems solved by technology

However, due to the different PCR amplification conditions for the detection of 14 serotypes by the method established by Xiao et al.

Method used

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  • Fluorescence quantitative PCR (polymerase chain reaction) detection method for 14 serotypes of Uu (ureaplasma urealyticum)
  • Fluorescence quantitative PCR (polymerase chain reaction) detection method for 14 serotypes of Uu (ureaplasma urealyticum)
  • Fluorescence quantitative PCR (polymerase chain reaction) detection method for 14 serotypes of Uu (ureaplasma urealyticum)

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[0026] The present invention will be described in detail below in conjunction with examples.

[0027] Specimens: 220 cases of female cervical secretions for physical examination; 14 serotype positive controls: serotype 1-14 standard strains, from the Epidemiology Laboratory of Southeast University, and strains 1-14 correspond to the American Standard Strain Collection Center (ATCC) The numbers are 27813, 27814, 27815, 27816, 2781727818, 27819, 21618, 33175, 33699, 33695, 33696, 33698 and 33697. .

[0028] Uu was isolated and cultured using the Mycoplasma IST2 kit (bioMérieus, France) according to the instructions, and 106 cases were finally confirmed to be Uu positive, and the positive liquid culture medium was stored in a -80°C refrigerator for later use.

[0029] The proteinase K method is used for DNA extraction. The simple steps are: take 1 ml of Uu-positive culture solution, centrifuge (14800 rpm, centrifugal radius 100 mm) for 10 minutes, discard the supernatant; add 60...

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Abstract

The invention discloses a fluorescence quantitative PCR (polymerase chain reaction) detection method for 14 serotypes of Uu (ureaplasma urealyticum). According to the method, the 14 serotypes adopt 20 microliterreaction systems, thefinal concentration of primers and probes is 0.2 mu mol / L (mu M), the final concentration of magnesium ions is 2 mmol / L (mM) for a dye method and 3 mM for a probe method, 10 microliters of qPCRMasterMix is added for the dye method, and 10 microliters of quantitativePCRSuperMix-UDG is added for the probe method. According to the method, the 14 serotypes of the Uu are typed with a molecular biological method for the first time, defects that a traditional typing method is low in sensitivity and poor in repeatability are overcome, a methodology basis is laid for further study on thepathogenicity of the Uu at a serotype level in the future, and the method can be widely applied clinically.

Description

technical field [0001] The invention relates to the field of genes, in particular to a fluorescent quantitative PCR detection method for 14 serotypes of Ureaplasma urealyticum. Background technique [0002] Ureaplasma urealyticum (Uu) is a common symbiotic microorganism in the human genitourinary tract, but it is also associated with various diseases, such as non-gonococcal urethritis (cervicitis), endometritis, Chorioamnionitis, neonatal meningitis, pneumonia, etc. [0003] There are currently 14 known serotypes of Uu, and the 14 serotypes have recently been divided into two biological groups according to various criteria, namely Ureaplasma parvum (UPA), including serotypes 1, 3, 6, and 14 Ureaplasma urealyticum (UUR) includes the remaining 10 serotypes. [0004] For many years, people have been speculating whether the pathogenicity of Uu is related to a specific biological group or serotype. Although many studies have reported that UUR is more pathogenic than UPA, some s...

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/686C12Q2561/101C12Q2563/107
Inventor 谢鑫友张钧宋铁军黄珺
Owner ZHEJIANG UNIV
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