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

Abstract

The invention discloses a fluorescent quantitative PCR detection method for 14 serotypes of Ureaplasma urealyticum. The method uses a 20 microliter reaction system for all 14 serotypes, and the final concentrations of primers and probes are both 0.2 micromole per liter. (uM), the final concentration of magnesium ions is 2 millimoles per liter (mM) for the dye method, and 3 mM for the probe method. Add 10 microliters of qPCR MasterMix for the dye method, and 10 microliters of quantitative PCRSuperMix-UDG for the probe method. The present invention realizes the typing of 14 serotypes of Ureaplasma urealyticum by molecular biological method for the first time, solves the shortcomings of low sensitivity and poor repeatability of traditional typing methods, and provides a basis for further research on urealyticum urealyticum at the level of serotypes in the future The pathogenicity of the protozoa has laid a methodological foundation and can be widely used in clinical practice.

Description

A fluorescent quantitative PCR detection method for 14 serotypes of Ureaplasma urealyticum

technical field

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

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.

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.

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 scholars do not agree with this view. Therefore, the different pathogenicity of Uu is likely to be related to different serotypes rather than biological groups. An accurate and feasible typing method is required to speculate on the correlation between a certain type of disease and a specific serotype.

Traditional antibody-based typing methods include growth/metabolism inhibition test, enzyme-linked immunosorbent assay, western blotting, etc. Antisera are not only cumbersome to prepare, difficult to commercialize, but also have insufficient sensitivity and stability, especially when the same When the specimen contains two or more serotypes, the resolution and repeatability are poor.

In the gene-based Uu typing methods in recent years, most of the primers and probes are designed for the difference in the 5' end sequence of the multiple-band antigen (MBA) gene, combined with direct sequencing or restriction Endonuclease analysis, these tests can separate the 4 serotypes of UPA, but can only divide the 10 serotypes of UUR into several groups. Recently Xiao et al. (Xiao L, 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.) Fluorescence Quantitative PCR (FQ-PCR) was used to establish a method for Uu grouping and typing, which successfully separated 14 serotypes without cross-reaction. Compared with traditional PCR, fluorescent quantitative PCR method has the characteristics of quantitative, more sensitive, fast and not easy to be contaminated, and has been widely used in the analysis of gene expression level, qualitative and quantitative detection of pathogens, etc. However, because the method established by Xiao et al. has different PCR amplification conditions for detecting 14 serotypes, it is very time-consuming and cumbersome in actual operation, so it is difficult to be widely used in clinical practice.

Contents of the invention

The object of the present invention is to aim at the deficiencies in the prior art, provide a kind of fluorescent quantitative PCR detection method of 14 kinds of serotypes of U. Designing some primers and probes, optimizing experimental conditions, and establishing an accurate and feasible fluorescent quantitative PCR detection method for Uu14 serotypes are of great significance to the research and clinical application of Uu pathogenicity.

The purpose of the present invention is achieved through the following technical solutions:

Proteinase K lysate: Mix 5.0ml KCl (1mol/L), 0.25ml MgCl ₂ (1mol/L), 1.5ml Tris-HCl (1mol/L, pH8.0), Tween-20 (20%) The lysate, lysate and proteinase K (200μg/ml) are mixed at a volume of 50:2, which is the proteinase K lysate used for DNA template extraction.

Fluorescent quantitative PCR was completed on Roche Light Cycler480 instrument. Among them, 9 serotypes 1, 3, 6, 14, 2, 7, 8, 9, and 13 were detected by the SYBR dye method, and the remaining 5 were detected by the Taqman probe method.

The kits used for SYBR dye method and probe method are Promega GoTaq qPCR Master Mix and Invitrogen quantitative PCR SuperMix-UDG, respectively.

All 14 serotypes use a 20 microliter reaction system, in which the final concentration of primers and probes is 0.2 micromoles per liter (uM), the final concentration of magnesium ions is 2 millimoles per liter (mM) for the dye method, and 2 millimoles per liter (mM) for the probe method For the dye method, add 10 microliters of Promega GoTaq qPCR Master Mix, and for the probe method, add 10 microliters of Invitrogen quantitative PCR SuperMix-UDG.

The conditions of the dye method PCR include a pre-warming at 95°C for 5 minutes, followed by 40 cycles of amplification. The conditions of the melting curve are: 95°C for 5 seconds (the rate is 4.4°C/s), 65°C for 1 minute (the rate is 2.2°C /s), 97°C (0.11°C/s) continuous acquisition signal mode. The final instrument cooling program was 40°C for 30 seconds. Conditions for the probe method included pre-warming at 50°C and 95°C for 2 minutes each, followed by an amplification procedure. The final instrument cooling program was 40°C for 30 seconds. Among them, serotypes 5 and 8 used Touch Down PCR to improve specificity. See the table below for detailed PCR conditions.

The standard strains of 14 serotypes are used as positive controls of Uu14 serotypes. The strains 1-14 correspond to the American Standard Strain Collection Center (ATCC) numbers 27813, 27814, 27815, 27816, 2781727818, 27819, 21618, 33175, 33699, 33695, 33696, 33698, and 33697. Sterile double distilled water was used as negative control.

Specific primers and/or Taqman probes for each serotype are listed in the table below

F is the upstream primer; R is the downstream primer; P is the probe (the 5' end of the probe is labeled with the reporter group FAM, and the 3' end is labeled with the quencher group TAMRA)

Ureaplasma urealyticum ATCC14 serotype standard strains were used to verify its specificity. The DNA concentration (ng/μl) of the purified standard strain was measured with a spectrophotometer NanoDrop2000C (purchased from Thermo Scientific Company), and the molecular copy number (unit/μl)=DNA mass concentration/DNA molecular weight=ng number× 6.02×10 ^14/ DNA molecular weight, DNA molecular weight=DNA base number×649, because only serotype 3 and 10 of the 14 serotypes have completed the whole genome sequencing, so the DNA base number UPA is based on serotype 3 (0.75-Mb) To calculate, UUR is calculated according to serotype 10 (0.87-Mb). Fluorescent quantitative PCR was performed after 10-fold serial dilution, and the amplification curve within 35 cycles was artificially regarded as positive, and the copy number of the lowest dilution that could yield a positive result within 35 cycles was the sensitivity of the test. The limit of detection of the assay ranged from 22 copies/µl (serotype 1) to 3200 copies/µl (serotype 5).

The beneficial effects of the present invention are that, through the design of primers and probes and repeated tests, the present invention detects 14 serotypes of Ureaplasma urealyticum by the fluorescent quantitative PCR method for the first time, and optimizes and unifies PCR conditions for some serotypes , especially the fluorescent quantitative PCR reaction conditions for the four serotypes of UPA (serotypes 1, 3, 6, and 14) are completely unified, which greatly facilitates the actual operation.

Description of drawings

Fig. 1 is the specific fluorescent quantitative PCR standard curve diagram of serotype 1;

In Figure 2, (a), (b), (c), and (d) are the specific amplification curves of 14 serotype standard strains detected by serotype 1, 3, 6, and 14-specific fluorescent quantitative PCR;

Figure 3 is the serotype distribution diagram of Ureaplasma urealyticum in female cervix, in which (a) is the result diagram of UPA serotype classification, and (b) is the result diagram of UUR serotype classification.

Detailed ways

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

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. .

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.

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 μL of proteinase K lysate to the precipitate, pipette Mix well; digest at 55°C for 60 minutes, inactivate at 95°C for 5 minutes; centrifuge for 1 minute, and then purify the supernatant with QIAamp DNA Mini Kit (purchased from QIAGEN) (operate according to the instructions) and then it will be the template for PCR amplification. Templates were stored in a -20°C refrigerator for later use.

Prepare proteinase K lysate for yourself: Mix 5.0ml KCl (1mol/L), 0.25ml MgCl2 (1mol/L), 1.5ml Tris-HCl (1mol/L, pH8.0), Tween-20 (20%) It is the lysate. After the lysate and proteinase K (200μg/ml) are mixed at a volume of 50:2, it is the proteinase K lysate used for DNA template extraction.

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.) The clinical Uu-positive strains were divided into two biogroups, namely UPA and UUR, by common PCR method, and then UPA was divided into 4 serotypes and UUR was divided into 10 serotypes according to the above-mentioned established fluorescent quantitative PCR method.

Fluorescent quantitative PCR reaction solution: 14 serotypes all use 20 microliters of reaction system, in which the final concentration of primers and probes is 0.2 micromole per liter (uM), and the final concentration of magnesium ion dye method is 2 millimole per liter ( mM), probe method is 3mM, dye method plus 10 microliters Promega GoTaq qPCR Master Mix, probe method plus 10 microliters Invitrogen quantitative PCR SuperMix-UDG.

Fluorescent quantitative PCR was completed on Roche Light Cycler480 instrument. Among them, 9 serotypes 1, 3, 6, 14, 2, 7, 8, 9, and 13 were detected by the SYBR dye method, and the remaining 5 were detected by the Taqman probe method. The conditions of the dye method PCR include a pre-warming at 95°C for 5 minutes, followed by 40 cycles of amplification. The conditions of the melting curve are: 95°C for 5 seconds (the rate is 4.4°C/s), 65°C for 1 minute (the rate is 2.2°C /s), 97°C (0.11°C/s) continuous acquisition signal mode. The final instrument cooling program was 40°C for 30 seconds. Conditions for the probe method included pre-warming at 50°C and 95°C for 2 minutes each, followed by an amplification procedure. The final instrument cooling program was 40°C for 30 seconds. See the table below for detailed PCR conditions.

Each assay included a positive control (corresponding standard strain) and a negative control (water replacement template).

The specific primers and probes for each serotype are shown in the table below.

Interpretation of the results, if there is a specific amplification curve within 35 cycles, it is considered positive. The specific classification results are shown in Figure 3.

<110> Zhejiang University

<120> A fluorescent quantitative PCR detection method for 14 serotypes of Ureaplasma urealyticum

<160> 33

<170> PatentIn version 3.3

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Claims (1)

1. a fluorescent quantitative PCR detection method for 14 kinds of serotypes of Ureaplasma urealyticum, is characterized in that, the method comprises: (1) All 14 serotypes use a 20 microliter reaction system, in which the final concentration of primers and probes is 0.2 micromole per liter (uM), and the final concentration of magnesium ion dye method is 2 millimole per liter (mM), Probe method is 3mM, dye method plus 10 microliters qPCR Master Mix, probe method plus 10 microliters quantitative PCR SuperMix-UDG; primers and labeled probes are as follows: Serum Type Primer, Probe Sequence (5’—3’) (2) Amplification conditions for the detection of 14 serotypes of Ureaplasma urealyticum by fluorescent quantitative PCR: