CN115807062A

CN115807062A - Primer combination for detecting mycoplasma pneumoniae by combining multi-cross isothermal amplification with nano biosensing and method for detecting mycoplasma pneumoniae

Info

Publication number: CN115807062A
Application number: CN202211306688.7A
Authority: CN
Inventors: 申阿东; 王亚翠; 焦伟伟
Original assignee: Beijing Childrens Hospital
Current assignee: Beijing Childrens Hospital
Priority date: 2019-03-19
Filing date: 2019-03-19
Publication date: 2023-03-17
Also published as: CN109929912A

Abstract

The invention discloses a primer combination for detecting mycoplasma pneumoniae by combining multi-cross isothermal amplification with nano biosensing, which can perform specific amplification on mycoplasma pneumoniae P1 genes. The invention also discloses a method for detecting mycoplasma pneumoniae by combining multi-cross isothermal amplification with nano biosensing, which can complete the whole MCDA amplification within 30 minutes, has the lower detection limit of 10fg in the detection of specific gene P1 of mycoplasma pneumoniae and has very high sensitivity. In addition, the amplification method provided by the invention has high amplification specificity. In the detection of common pathogenic bacteria, conditional pathogenic bacteria and non-mycoplasma pneumoniae DNA, the method is negative amplification, and only the mycoplasma pneumoniae is specifically amplified, so that the established method has extremely high specificity, can accurately identify the mycoplasma pneumoniae, and has no false positive and false negative results.

Description

Primer combination for detecting mycoplasma pneumoniae by combining multi-cross isothermal amplification with nano biosensing and method for detecting mycoplasma pneumoniae

The application is a divisional application with the application date of 2019, 03 and 19 months, the application number of 201910209767.8 and the invention name of a method for detecting mycoplasma pneumoniae by combining multi-crossover isothermal amplification with nano biosensing.

Technical Field

The invention belongs to the technical field of molecular biology, and discloses a primer combination for detecting mycoplasma pneumoniae by combining multi-cross isothermal amplification with nano biosensing and a method for detecting mycoplasma pneumoniae.

Background

Mycoplasma Pneumoniae (MP) is one of the most common pathogens of human respiratory infectious diseases and is also the main pathogen of community-acquired pneumonia. In the MP epidemic period, community-acquired pneumonia caused by MP accounts for about 20% -40% in the general population and up to 70% in the closed population. MP infections can occur in people of all ages, with children and adolescents in school age being the most common. Clinical symptoms caused by MP infection are various, and the clinical manifestations of mild patients are similar to those of nonspecific symptoms caused by other pathogen infections, such as fever, cough, watery nasal discharge and the like; severe cases may manifest as severe respiratory diseases such as severe pneumonia and respiratory failure, even affecting the central system and digestive system. Beta-lactam drugs act on cell walls and are often selected drugs for empirical treatment of respiratory infectious diseases, however, MP lacks cell walls and is naturally resistant to drugs acting on the cell walls. Therefore, the development of a simple, rapid, sensitive and specific MP detection method for diagnosing MP infection is crucial to the rational application of antibiotics and the effective treatment of MP.

The current MP detection methods for diagnosis of MP infection are mainly: culture method, serological detection and molecular biological detection. The culture method is the gold standard for MP detection, but the method is not recommended to be used for MP detection because the requirements of MP on growth conditions are severe, laboratories have high requirements, technicians have high requirements, and MP grows slowly, and the culture result can be obtained in 2-4 weeks. Serological detection is widely used for laboratory detection due to good convenience, sensitivity and specificity, but is influenced by sampling time, age of a patient, immune state of an organism and other factors, meanwhile, the reliability of a serological detection result depends on the acquisition of double serum in an acute stage and a recovery stage, and the serological detection is limited in clinical application due to the fact that the serum in the recovery stage is difficult to acquire. With the development of molecular biology technology, nucleic acid detection technology based on PCR is beginning to be widely used for MP detection, wherein Real-time PCR is the MP rapid detection technology commonly used in clinic at present. However, the detection method depends on a precise and complicated instrument and an expensive probe, and has high requirements on laboratory conditions and technicians, so that the detection method is not suitable for primary hospitals.

In recent years, nucleic acid detection techniques based on isothermal amplification techniques have been rapidly developed to overcome the deficiencies of PCR detection. Compared with the PCR technology, the isothermal amplification technology does not depend on thermal cycle amplification equipment, only needs a water bath or a simple PCR instrument, and has the advantages of high specificity and sensitivity, simple operation and rapid reaction, so the isothermal amplification technology can be widely applied to various medical institutions. Up to now, more than 10 kinds of isothermal amplification techniques have been used, and currently, rolling Circle Amplification (RCA), strand Displacement Amplification (SDA), helicase-dependent isothermal amplification (HDA), loop-mediated isothermal amplification (LAMP), cross amplification (CPA), and the like are widely used. However, these isothermal amplification techniques rely on multiple enzymes to work simultaneously, and the reagents are expensive and complex to operate, and are not suitable for popularization and application in less-developed areas and in the field of rapid detection techniques. In order to overcome the disadvantages of the PCR technology and the existing isothermal amplification technology and to realize simple, rapid, sensitive and specific nucleic acid detection, the inventors recently created a new nucleic acid amplification technology named Multiple Cross Displacement Amplification (MCDA), the related content of which is disclosed in CN104946744A, which is a part of the specification of the present application as a prior art document.

Under the condition of constant temperature, MCDA only uses a constant temperature substitution enzyme to realize nucleic acid amplification, and has rapid reaction and high sensitivity and specificity. In order to make the technology more economical and be popularized and applied in the fields of biology, medicine and health, the inventor takes MCDA as the basis, combines the MCDA technology with the nano-biological detection technology, develops the nano-biological sensing technology which depends on the MCDA technology and realizes quick and sensitive detection, is named as a multi-crossover Cross constant temperature Amplification and nano-biological sensing combined nucleic acid detection technology (MCDA-LFB), and related contents are disclosed in CN201610872509.4, CN201610942289.8 and CN201610982015.1, and the patent documents serve as part of the prior art document forming the specification of the application. How to apply MCDA-LFB to MP detection, and designing MCDA amplification primers aiming at specific genes P1 of MP to quickly, sensitively and specifically detect MP still remains the problem to be mainly solved by the industry.

Disclosure of Invention

The invention aims to apply MCDA-LFB to MP detection, design MCDA amplification primers aiming at specific genes P1 of MP, and establish a rapid, sensitive and specific MCDA-LFB detection system aiming at MP.

Based on the above purpose, the present invention firstly provides a method for detecting a target gene by combining multi-cross isothermal amplification with nano biosensing, which comprises the following steps:

(1) Extracting a genome of a sample to be detected;

(2) Providing a replacement primer F1 with a sequence shown as SEQ ID NO. 1 and a replacement primer F2 with a sequence shown as SEQ ID NO. 2, providing a cross primer CP1 with a sequence shown as SEQ ID NO. 3 and a cross primer CP2 with a sequence shown as SEQ ID NO. 4; an amplification primer C1 with a sequence shown as SEQ ID NO. 5 and an amplification primer C2 with a sequence shown as SEQ ID NO. 6, an amplification primer D1 with a sequence shown as SEQ ID NO. 7 and an amplification primer D2 with a sequence shown as SEQ ID NO. 8, an amplification primer R1 with a sequence shown as SEQ ID NO. 9 and an amplification primer R2 with a sequence shown as SEQ ID NO. 10; wherein hapten is marked at the 5 'end of the primer C1 or C2, and biotin is marked at the 5' end of the primer D1 or D2; the primer designed in the step is designed aiming at the specific gene P1 of the mycoplasma pneumoniae;

(3) Under the existence of chain-shifting polymerase, a melting temperature regulator and a primer, using the genome nucleic acid of a sample to be detected as a template to amplify DNA at constant temperature; the amplification in the step is carried out by taking the nucleic acid sequence of the specific gene P1 of the mycoplasma pneumoniae as a template;

(4) Detecting the amplification product of step (3) by using a nano biosensor.

In a preferred embodiment, the primer labeled with hapten at the 5 'end is C1, the primer labeled with hapten is defined as C1, the primer labeled with biotin at the 5' end is D1, and the primer labeled with biotin is defined as D1.

In a more preferred embodiment, the hapten labeled at the 5' end of the amplification primer C1 is Fluorescein (FITC).

More preferably, the nano biosensor comprises a back plate, wherein a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the back plate, a detection line and a control line are sequentially arranged on the nitrocellulose membrane, and the binding pad, the detection line and the control line are sequentially coated with the colored group modified avidin-based polymer nanoparticles, the goat anti-FITC antibody and the biotin-coupled bovine serum albumin.

In another preferred embodiment, the isothermal amplification is performed at a temperature of 64-66 ℃.

More preferably, the isothermal amplification is performed in an environment at 65 ℃.

In another preferred embodiment, the isothermal amplification time is 30 minutes.

Secondly, the invention also provides a group of primers for isothermal amplification of P1 gene of mycoplasma pneumoniae, wherein the primers comprise: a displacing primer F1 with a sequence shown as SEQ ID NO. 1, a displacing primer F2 with a sequence shown as SEQ ID NO. 2, a cross primer CP1 with a sequence shown as SEQ ID NO. 3 and a cross primer CP2 with a sequence shown as SEQ ID NO. 4; an amplification primer C1 with a sequence shown as SEQ ID NO. 5 and an amplification primer C2 with a sequence shown as SEQ ID NO. 6, an amplification primer D1 with a sequence shown as SEQ ID NO. 7 and an amplification primer D2 with a sequence shown as SEQ ID NO. 8, an amplification primer R1 with a sequence shown as SEQ ID NO. 9 and an amplification primer R2 with a sequence shown as SEQ ID NO. 10; wherein hapten is marked at the 5 'end of the primer C1 or C2, and biotin is marked at the 5' end of the primer D1 or D2.

The multi-cross displacement amplification method disclosed by the invention can complete the whole MCDA amplification within 30 minutes, and has a very high sensitivity, wherein the lower detection limit can be as low as 10fg in the detection of the specific gene P1 of mycoplasma pneumoniae. In addition, the amplification method provided by the invention has high amplification specificity. In the detection of common pathogenic bacteria, conditional pathogenic bacteria and non-mycoplasma pneumoniae DNA, the method is negative amplification, and only the mycoplasma pneumoniae is specifically amplified, which shows that the established method has extremely high specificity, can accurately identify the mycoplasma pneumoniae and does not generate false positive and false negative results.

Drawings

FIG. 1 is a schematic diagram showing the position and orientation of MCDA-LFB primer design;

FIG. 2 is a diagram of the results of MCDA primer verification and MCDA-LFB detection;

FIGS. 3-1 and 3-2 are graphs of the results of the test for the optimum reaction temperature of a standard MCDA-LFB;

FIG. 4 is a graph showing the sensitivity results of MCDA-LFB detection of Mycoplasma pneumoniae;

FIG. 5 is a specific detection evaluation map of MCDA-LFB technology;

FIG. 6 is a graph of the results of an optimal response time test for the MCDA-LFB technique.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.

Materials and methods

1. Reagents involved in the invention:

anti-fluorescein antibody (anti-FITC) and biotin-conjugated bovine serum albumin (B-BSA) were purchased from Abcam. Polymeric nanoparticle-coupled streptavidin (SA-NP) was purchased from Beijing Haitangyuan technologies, inc. The backing sheet, sample pad, conjugate pad, absorbent pad and fibrous membrane were purchased from shanghai jeikei. Isothermal amplification Kit (amplification Kit) and isothermal amplification visible dye (VR) were purchased from tianjin jieyaite biotechnology limited. DNA extraction kits (QIAamp DNA minikits; qiagen, hilden, germany) were purchased from Qiagen, germany. The other reagents are all commercial parting pure products.

The main instruments used in the experiment of the invention: constant temperature real time turbidimeter LA-320C (Eiken Chemical Co., ltd, japan) was purchased from Japan Rongyan Co. The PCR instrument is Dong Sheng Long EDC-810, beijing Dong Sheng Innovation Biotechnology Co.

Genome extraction: the bacterial genome was extracted using a DNA extraction kit from Qiagen (QIAamp DNAminiks; qiagen, hilden, germany) according to the instructions. The extracted genome DNA is subpackaged in small quantity and stored at-20 ℃ for later use.

2. Primer design

In order to verify and evaluate the MCDA-LFB technology and establish a rapid, sensitive and specific MCDA-LFB detection system aiming at MP. The invention designs a set of MCDA amplification primers aiming at specific gene P1 of MP, and aims to verify the feasibility, sensitivity, specificity and reliability of the MCDA-LFB technology. The P1 gene exists in all MP, and can distinguish MP from other strains due to good conservation and high specificity. MCDA primers are designed by using Primer design software PrimeExplorer V4 (Eiken Chemical) (http:// PrimeExplorer. Jp/e /) and Primer design software Primer Premier 5.0, and the designed MP specific primers are subjected to sequence alignment in NCBI database (http:// blast. NCBI. Nlm. Nih. Gov/blast. Cgi), so that non-specific matching of the primers and other species sequences can exist is eliminated, and finally, the optimized MCDA amplification primers are obtained. The primer design is schematically shown in FIG. 1. Primer sequences and modifications are shown in table 1.

TABLE 1 primer sequences and modifications

C1 is a primer labeled with FITC (fluorescein) at the 5' end, which is used in the MCDA-LFB detection system);

d1 is a primer labeled with Biotin (Biotin) at the 5' end, which is used in the MCDA-LFB detection system).

3. Biological sensor detector (LFB)

Detection principle of LFB: the MP-MCDA amplification product was added dropwise directly to the LFB pad area, and then 80. Mu.L of detection buffer was added to the sample pad area, and the MCDA product was wicked from bottom to top (from the sample pad to the absorbent pad). When the MCDA product reaches the binding pad, one end of the ditag product (i.e., the biotin-labeled end) reacts with SA-NP (nanoparticle-coupled streptavidin). When the product continues to move, the other end (i.e. the fluorescein labeled end) of the dual-standard product is combined with the antibody of the detection line area, and the dual-standard product is fixed in the detection line area. As the products accumulate in the detection line region, the color reaction is performed through the SA-NP at the other end, so that the MCDA products are visually detected. In addition, excess SA-NP was directly reacted with B-BSA (Biotin-coupled bovine serum albumin) in the CL (quality control line) region to determine whether or not the LFB function was normal.

LFB result reading (fig. 2): a positive result (B1 in fig. 2) is represented by red bands appearing in both the CL (quality control line) region and the TL (detection line) region; a negative result (B2-4 in fig. 2) was represented by red bands appearing only in the CL region; when the LFB test strip does not have a red strip, the LFB test is invalid; when only the TL area has a red strip and the CL area has no red strip, the detection result is unreliable and needs to be detected again.

Example 1 feasibility verification of MCDA primers

MCDA amplification (see in detail CN 201610982015.1)

MCDA reaction system: 0.4 μ M displacement primers F1 and F2,0.8 μ M amplification primers C1 and C2, 1.2 μ M amplification primers D1, D2, R1, R2,2.4 μ M crossover primers CP1 and CP 2. 10mM Betain, 6mM MgSO ₄ 1mM dNTP, 12.5. Mu.L of 10 XBstDNA polymerase buffer, 10U of strand displacement DNA polymerase, 1. Mu.L of template, 1. Mu.L of LVR dye, and 25. Mu.L of deionized water. The MCDA is reacted at a constant temperature of 65 ℃ for 40min.

After the amplification of MCDA, there are two detection methods that can be used for discrimination of the MCDA amplification product, one is that by adding a visible dye (VR) to the reaction mixture, the color of the positive reaction tube is green (A1 in FIG. 2), and the negative reaction is colorless (A2-4 in FIG. 2). Another most straightforward method is to test the product by LFB, with positive results showing red bands in both the CL and TL regions (B1 in FIG. 2) and negative results showing only red bands in the CL region (B2-4 in FIG. 2).

Example 2 determination of optimum reaction temperature for MCDA technology

Under the condition of a standard MCDA reaction system, an MPDNA template and a designed MCDA primer are added, and the concentration of the MP template is 1 ng/. Mu.L. The reaction was carried out at constant temperature (61-68 deg.C) and different dynamic curves were obtained at different temperatures using real-time turbidimeter measurements, see FIGS. 3-1 and 3-2. 64-66 ℃ is recommended as the optimal reaction temperature for MCDA primers. The subsequent verification in the invention selects 65 ℃ as a constant temperature condition for MCDA amplification. FIGS. 3-1 and 3-2 show the dynamic graphs of the screening temperature of the MP-detecting MCDA primers designed for the P1 gene sequence.

Example 3 MP-MCDA sensitivity to MP

After MP standard strain genome DNA (1 ng/. Mu.L) is diluted in a continuous multiple ratio, standard MCDA amplification is carried out, and the amplification product of MCDA is detected by LFB, and the results show that: the MP genomic DNA concentration range was 1ng-10fg by MCDA-LFB detection, and a red band appeared in both CL and TL regions by LFB, which was a positive result (B1-B6 in FIG. 4). When the concentration of MP genomic DNA decreased below 10fg, LFB appeared as a red band only in the CL region, which was a negative result (B7-B8 in FIG. 4). In B1-B7 in FIG. 4, the MP template concentrations are 1ng, 100pg, 10pg, 1pg,100fg,10fg, and 1fg in this order, and B8 in FIG. 4 is a blank control. To further verify the sensitivity of MCDA-LFB to MP, a visible dye (VR) was added to the standard reaction system, and the MP positive reaction tubes appeared green and the MP negative reaction tubes changed from green to colorless. The detection result of the visible dye (VR) shows that: MP-MCDA was detected in the range of 1ng to 10fg, the color of the positive amplification tube was green (A1-A6 in FIG. 4), and when the amount of MP genomic DNA in the reaction system decreased to 10fg or less, the color of the reaction tube changed from green to colorless, indicating a negative result (A7-A8 in FIG. 4). In FIG. 4, the concentrations of A1-A7 and MP templates were 1ng, 100pg, 10pg, 1pg,100fg,10fg, and 1fg, respectively, and FIG. 4A8 is a blank control.

Example 4 detection of MP specificity by MCDA-LFB technique

The specificity of the MP-MCDA-LFB technology (the strain information is shown in the detailed table 2) is evaluated by taking common pathogenic bacteria DNA (streptococcus pneumoniae, staphylococcus aureus, staphylococcus epidermidis, staphylococcus saprophyticus, klebsiella pneumoniae, pseudomonas aeruginosa, campylobacter jejuni, escherichia coli, shigella, shigella bodyii, listeria monocytogenes, bacillus cereus, enterobacter sakazakii, aeromonas hydrophila, vibrio parahaemolyticus, vibrio vulnificus, enterococcus pauci, enterococcus faecalis and enterococcus faecium) as a template, and the technology can accurately identify MP and other pathogens and has good specificity, and is shown in the figure 5. In FIG. 5, LFB1-6: an MPDNA template; LFB7-27: other pathogenic DNA templates; LFB28: blank control. The result shows that the MP-MCDA-LFB technology can accurately detect the MP target sequence.

TABLE 2 strains and results of specificity detection

BCH, beijing Children's Hospital; CDC, CHEnese Center for disease Control and preservation (Chinese Center for disease Prevention Control), P, positive (MP-MCDA-LFB detection positive); the detection results in table 2 show that only the members belonging to m.pnemoniae generate positive results through MP-MCDA-LFB detection, and the established method accurately identifies m.pnemonia and generates no false positive and false negative results.

Example 5 determination of the optimal reaction time of the MP-MCDA-LFB technique

Under standard MCDA amplification reaction conditions, an MP genomic DNA template and a corresponding MCDA primer designed for the P1 gene are added. The reaction was carried out at a constant temperature of 65 ℃ for 10 minutes, 20 minutes, 30 minutes and 40 minutes in this order. LFB detection results show that: the optimum reaction time for the application of the MCDA-LFB technique to MP detection is 30 minutes (FIG. 6). When the MCDA isothermal amplification time was controlled to 30 minutes, all templates at the detection line level could be detected (C in fig. 6). In C of FIG. 6, LFB detection ranged from 1pg to 10fg, and red bands appeared in both TL and CL regions in LFB (C1-C3 in FIG. 6). When the amount of the genomic template in the reaction system decreased to 10fg or less, LFB appeared as a red line only in the CL region, indicating a negative result (C4 in FIG. 6). FIG. 6 shows the amplification results of the MCDA system using LFB reading from 10 minutes to 40 minutes; the template amounts of MP represented by C1 to C4 were 1Pg,100fg,10fg,1fg, C5 and C6, respectively, as a negative control (Streptococcus pneumoniae template 1 pg) and a blank control (1. Mu.L double distilled water).

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and all of the embodiments are included in the scope of the present invention.

Claims

1. A primer combination for detecting mycoplasma pneumoniae by combining multi-cross isothermal amplification with nano biosensing is characterized by comprising a displacement primer, a cross primer and an amplification primer;

the replacement primer comprises a replacement primer F1 with a sequence shown as SEQ ID NO. 1 and a replacement primer F2 with a sequence shown as SEQ ID NO. 2;

the cross primer comprises a cross primer CP1 with a sequence shown as SEQ ID NO. 3 and a cross primer CP2 with a sequence shown as SEQ ID NO. 4;

the amplification primer comprises an amplification primer C1 with a sequence shown as SEQ ID NO. 5, an amplification primer C2 with a sequence shown as SEQ ID NO. 6, an amplification primer D1 with a sequence shown as SEQ ID NO. 7, an amplification primer D2 with a sequence shown as SEQ ID NO. 8, an amplification primer R1 with a sequence shown as SEQ ID NO. 9 and an amplification primer R2 with a sequence shown as SEQ ID NO. 10; wherein hapten is marked at the 5 'end of the amplification primer C1 or C2, and biotin is marked at the 5' end of the amplification primer D1 or D2.

2. The amplification primer according to claim 1, wherein hapten is labeled at the 5' end of the amplification primer C1, and the primer labeled with hapten is defined as an amplification primer C1; biotin is marked at the 5' end of the amplification primer D1, and the primer marked with biotin is designated as an amplification primer D1.

3. The amplification primer of claim 2, wherein the hapten labeled at the 5' end of the amplification primer C1 is fluorescein.

4. Use of a primer combination according to any one of claims 1 to 3 for the preparation of a kit for the detection of mycoplasma pneumoniae.

5. A method for detecting a target gene based on multi-crossover isothermal amplification combined with nano-biosensing for non-diagnostic purposes, the method comprising the steps of:

(1) Extracting a genome of a sample to be detected;

(2) Providing a primer combination of claim 1;

(3) Under the existence of chain-shifting polymerase, a melting temperature regulator and the primer combination in the step (2), using the genome nucleic acid of the sample to be detected as a template for isothermal amplification of DNA;

(4) Detecting the amplification product of step (3) by using a nano biosensor.

6. The method of claim 5, wherein the nanobiosensor comprises a back plate, a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad are sequentially disposed on the back plate, a detection line and a control line are sequentially disposed on the nitrocellulose membrane, and the binding pad, the detection line and the control line are sequentially coated with the colored group modified avidin high molecular nanoparticles, goat anti-FITC antibody and biotin conjugated bovine serum albumin.

7. The method of claim 5, wherein the isothermal amplification is performed in an environment of 64-66 ℃.

8. The method of claim 7, wherein the isothermal amplification is performed in an environment at 65 ℃.

9. The method of claim 5, wherein the isothermal amplification time is 30 minutes.