CN103966315A

CN103966315A - Fluorescence quantitative PCR (polymerase chain reaction) detection method for 14 serotypes of Uu (ureaplasma urealyticum)

Info

Publication number: CN103966315A
Application number: CN201410113941.6A
Authority: CN
Inventors: 谢鑫友; 张钧; 宋铁军; 黄珺
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2014-03-25
Filing date: 2014-03-25
Publication date: 2014-08-06

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

14 kinds of serotype fluorescent quantitative PCR detection methods of a kind of ureaplasma urealyticum

Technical field

The present invention relates to gene field, relate in particular to 14 kinds of serotype fluorescent quantitative PCR detection methods of a kind of ureaplasma urealyticum.

Background technology

Ureaplasma urealyticum (Ureaplasma urealyticum, Uu) be a kind of common symbiotic type microorganism in mankind's urogenital tract, but simultaneously also relevant with various diseases, as non gonococcal urethritis (cervicitis), endometritis, chorioamnionitis, neonatal meningitis, pneumonia etc.

What Uu was at present known has 14 kinds of serotypes, nearest 14 kinds of serotypes are divided into two biological groups according to various standards again, be ureaplasma parvum (Ureaplasma parvum, UPA) comprise serotype 1,3,6,14 and ureaplasma urealyticum (Ureaplasma urealyticum, UUR) comprise all the other 10 kinds of serotypes.

People are guessing the pathogenic whether relevant with a certain specific biological group or serotype of Uu always for many years, pathogenic although a lot of research report UUR has more than UPA, also have scholar and disagree with this viewpoint.So the pathogenic probably instead of biological group relevant with different serotypes that Uu is different, infers that the dependency between a certain class disease and particular serotype just needs a kind of accurate, feasible classifying method.

Traditional is that basic classifying method comprises growth/metabolic inhibition test, enzyme linked immunosorbent assay, immunoblotting etc. based on antibody, that antiserum(antisera) is not only prepared is loaded down with trivial details, be difficult to commercialization, and susceptibility and stability are inadequate, particularly, in the time containing two kinds or above serotype in same sample, resolving power and repeatability are all poor.

In recent years taking gene in basic Uu classifying method, the design of primer and probe is to design for the difference of multi-ribbon antigen (multiple-band antigen MBA) gene 5 ' end sequence mostly, in conjunction with direct Sequencing or restriction endonuclease analysis, these tests can separate 4 of a UPA serotype type, but 10 of a UUR serotype can only be divided into several groups.(the Xiao L such as nearest Xiao, Glass J I, Paralanov V, et al.Detection and characterization of human Ureaplasma species and serovars by real-time PCR[J] .J Clin Microbiol, 2010,48 (8): 2715-2723.) with quantitative fluorescent PCR (Fluorescence Quantitative PCR, FQ-PCR) set up that a set of Uu hives off and the method for somatotype, success 14 kinds of serotypes separately and there is no a cross reaction.Compared with normal PCR fluorescence quantitative PCR method have can be quantitatively, sensitiveer, fast and be not easy contaminated feature, be widely used at aspects such as the qualitative and quantitative analyses of gene expression dose analysis, pathogenic agent.But it is different that the method for setting up due to Xiao etc. detects the pcr amplification condition of 14 kinds of serotypes, make in actually operating very consuming timely and loaded down with trivial details, be therefore difficult to extensively carry out in clinical.

Summary of the invention

The object of the invention is to for the deficiencies in the prior art, provide a kind of ureaplasma urealyticum 14 kinds of serotype fluorescent quantitative PCR detection methods, the present invention on the basis in conjunction with document by Uu14 kind serotype genome is analyzed, redesign part primer and probe optimization experiment condition, set up a set of accurate and practicable Uu14 kind serotype fluorescent quantitative PCR detection method, the studies and clinical application pathogenic to Uu all has very important significance.

The object of the invention is to be achieved through the following technical solutions:

Proteinase K lysate: get 5.0ml KCl(1mol/L), 0.25ml MgCl ₂(1mol/L), 1.5ml Tris-HCl(1mol/L, PH8.0), Tween-20(20%) mix be lysate, after lysate and Proteinase K (the 200 μ g/ml) volume mixture with 50:2, be DNA profiling and extract Proteinase K lysate used.

Quantitative fluorescent PCR completes on Roche Light Cycler480 instrument.Wherein serotype 1,3,6,14,2,7,8,9,13 totally 9 kinds be SYBR dye method, use Taqman probe method for all the other 5 kinds.

SYBR dye method and probe method test kit used is respectively Promega GoTaq qPCR Master Mix and Invitrogen quantitative PCR SuperMix-UDG.

14 kinds of serotypes all adopt 20 microlitre reaction systems, wherein the final concentration of primer and probe is every liter of 0.2 micromole (uM), magnesium ion final concentration dye method is every liter of 2 mmole (mM), probe method is 3mM, dye method adds 10 microlitre Promega GoTaq qPCR Master Mix, and probe method adds 10 microlitre Invitrogen quantitative PCR SuperMix-UDG.

The condition of dye method PCR all comprises 95 DEG C of pre-temperature of 5 minutes, then 40 cyclic amplifications, the condition of melting curve is: 95 DEG C 5 seconds (speed be 4.4 DEG C/s), 65 DEG C 1 minute (speed be 2.2 DEG C/s), 97 DEG C (speed is 0.11 DEG C/s) continuous acquisition signal mode.40 DEG C of last instrument cooling programs 30 seconds.The condition of probe method comprises 50 DEG C of pre-temperature and 95 DEG C each 2 minutes, then amplification program.40 DEG C of last instrument cooling programs 30 seconds.Wherein serotype 5,8 is in order to improve specificity landing (Touch Down) PCR.PCR condition sees the following form in detail.

Uu14 kind serotype mark positive control adopts the reference culture of 14 kinds of serotypes, and the corresponding USS bacterial strain preservation center of bacterial classification 1-14 type (ATCC) numbering is respectively 27813,27814,27815,27816,2781727818,27819,21618,33175,33699,33695,33696,33698 and 33697.Negative control adopts aseptic double-distilled water.

The special primer of each serotype and or Taqman probe see the following form

F is upstream primer; R is downstream primer; P is probe (probe 5 ' end mark reporter group FAM, 3 ' end mark quenching group TAMRA)

Verify its specificity with ureaplasma urealyticum ATCC14 kind serotype reference culture.DNA concentration (nanogram/microlitre) after reference culture purifying with spectrophotometer NanoDrop2000C(purchased from Thermo Scientific company) measure molecule copy number (individual/microlitre)=DNA mass concentration/DNA molecular amount=nanogram number × 6.02 × 10 ^14/dNA molecular amount, DNA molecular amount=DNA base number × 649, because 14 kinds of serotypes only have serotype 3 and 10 to complete genome sequencing, so DNA base is counted UPA by serotype 3(0.75-Mb) calculate, UUR calculates by serotype 10 (0.87-Mb).After becoming 10 times of gradient dilutions, carry out quantitative fluorescent PCR, artificially establish the positive that is considered as that has amplification curve in 35 circulations, in 35 circulations, can show that the minimum dilution copy number of positive findings is the susceptibility of this test.The detection limit scope of this test is that 22 copies/microlitre (serotype 1) is to 3200 copies/microlitre (serotype 5).

The invention has the beneficial effects as follows, the present invention is by the design to primer and probe and test repeatedly, utilize first fluorescence quantitative PCR method to detect 14 kinds of serotypes of ureaplasma urealyticum, for optimization and the unification of part serotype PCR condition, particularly to UPA(serotype 1,3,6,14) the quantitative fluorescent PCR reaction conditions of 4 serotypes unifies completely, greatly facilitates actual operation.

Brief description of the drawings

Fig. 1 is the special quantitative fluorescent PCR canonical plotting of serotype 1;

In Fig. 2, (a) and (b), (c), (d) are respectively the specific amplification curve of the special 14 kinds of serotype reference cultures of fluorescence quantitative PCR detection of serotype 1,3,6,14;

Fig. 3 is women's uterine cervix ureaplasma urealyticum serotype distribution figure, wherein, and (a) for UPA divides serotype result figure, (b) for UUR divides serotype result figure.

Embodiment

Below in conjunction with example, the present invention is described in detail.

Sample: health check-up women cervical secretions sample 220 examples; 14 kinds of serotype positive controls: serotype 1-14 reference culture, university's EL southeast, the corresponding USS bacterial strain preservation center of bacterial classification 1-14 type (ATCC) numbering is respectively 27813,27814,27815,27816,2781727818,27819,21618,33175,33699,33695,33696,33698 and 33697.。

The separation and Culture of Uu adopts Mycoplasma IST2 test kit (bioM é rieus, France) by specification to operate, and what finally confirm as the Uu positive is 106 examples, for subsequent use in DEG C refrigerator of positive liquid medium-80.

The extraction of DNA adopts Proteinase K method, and easy steps is: get 1 milliliter of the positive nutrient solution of Uu, centrifugal (14800 revs/min, 100 millimeters of centrifugal radiuses) 10 minutes, abandon supernatant liquor; In precipitation, add Proteinase K lysate 60 μ L, piping and druming mixes; 55 DEG C of digestion 60 minutes, 95 DEG C of deactivations 5 minutes; Centrifugal 1 minute, supernatant liquor was used QIAamp DNA Mini Kit(again purchased from QIAGEN company) be pcr amplification template after purifying (by specification operation).Template is kept in-20 DEG C of refrigerators for subsequent use.

Proteinase K lysate is prepared for own: get 5.0ml KCl(1mol/L), 0.25ml MgCl2(1mol/L), 1.5ml Tris-HCl(1mol/L, PH8.0), Tween-20(20%) mix and be lysate, after lysate and Proteinase K (the 200 μ g/ml) volume mixture with 50:2, be DNA profiling and extract Proteinase K lysate used.

First reference (Teng L J, Zheng X, Glass J I, et al.Ureaplasma urealyticum biovar specificity and diversity are encoded in multiple-banded antigen gene[J] .J Clin Microbiol, 1994,32 (6): 1464-1469.) by regular-PCR method, clinical Uu positive strain is divided into two biological groups, be UPA and UUR, and then according to the above-mentioned fluorescence quantitative PCR method of having set up, UPA be divided into 4 serotypes, UUR and be divided into 10 serotypes.

Fluorescence quantitative PCR reaction solution: 14 kinds of serotypes all adopt 20 microlitre reaction systems, wherein the final concentration of primer and probe is every liter of 0.2 micromole (uM), magnesium ion final concentration dye method is every liter of 2 mmole (mM), probe method is 3mM, dye method adds 10 microlitre Promega GoTaq qPCR Master Mix, and probe method adds 10 microlitre Invitrogen quantitative PCR SuperMix-UDG.

Quantitative fluorescent PCR completes on Roche Light Cycler480 instrument.Wherein detect serotype 1,3,6,14,2,7,8,9,13 totally 9 kinds be SYBR dye method, use Taqman probe method for all the other 5 kinds.The condition of dye method PCR all comprises 95 DEG C of pre-temperature of 5 minutes, then 40 cyclic amplifications, the condition of melting curve is: 95 DEG C 5 seconds (speed be 4.4 DEG C/s), 65 DEG C 1 minute (speed be 2.2 DEG C/s), 97 DEG C (speed is 0.11 DEG C/s) continuous acquisition signal mode.40 DEG C of last instrument cooling programs 30 seconds.The condition of probe method comprises 50 DEG C of pre-temperature and 95 DEG C each 2 minutes, then amplification program.40 DEG C of last instrument cooling programs 30 seconds.PCR condition sees the following form in detail.

Each test all comprises positive control (corresponding reference culture) and negative control (water alternate template).

Special primer, the probe of each serotype sees the following form.

Result interpretation, has the positive that is considered as of specific amplification curve in 35 circulations.Concrete somatotype result as shown in Figure 3.

<110> Zhejiang University

14 kinds of serotype fluorescent quantitative PCR detection methods of <120> ureaplasma urealyticum

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<170> PatentIn version 3.3

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Claims

1. 14 kinds of serotype fluorescent quantitative PCR detection methods of ureaplasma urealyticum, is characterized in that, the method comprises:

(1) 14 kind of serotype all adopts 20 microlitre reaction systems, wherein the final concentration of primer and probe is every liter of 0.2 micromole (uM), magnesium ion final concentration dye method is every liter of 2 mmole (mM), probe method is 3mM, dye method adds 10 microlitre qPCR Master Mix, and probe method adds 10 microlitre quantitative PCR SuperMix-UDG; Primer and label probe are as follows:

Serotype primer, probe sequence (5 '-3 ')

(2) 14 kinds of serotype amplification conditions of fluorescence quantitative PCR detection ureaplasma urealyticum: