Mycoplasma pneumoniae gene detection kit based on silicon-based micro-fluidic chip and detection method thereof

Abstract

The invention discloses a mycoplasma pneumoniae gene detection kit based on a silicon-based micro-fluidic chip and a detection method thereof, and the kit provided by the invention comprises a rapid hot start DNA polymerase, a PCR reaction mixed solution, a positive reference substance and a negative reference substance, and also comprises a PCR reaction system which takes a mycoplasma pneumoniae repMp1 gene as a target gene, the following primers are designed by a PCR (Polymerase Chain Reaction) fluorescent probe method: an upstream primer F, a downstream primer R and a detection probe P, and the nucleotide sequences of the upstream primer F, the downstream primer R and the detection probe P are shown as SEQ ID NO.1-3. The mycoplasma pneumoniae gene detection method provided by the invention comprises the steps of extraction of pathogen DNA (Deoxyribonucleic Acid) and PCR (Polymerase Chain Reaction) amplification detection. According to the invention, the mycoplasma pneumoniae gene detection kit which is more comprehensive in detection effect, high in specificity, good in sensitivity, low in omission ratio, convenient, simple and easy to operate is provided.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnostic reagents, and in particular to a Mycoplasma pneumoniae gene detection kit based on a silicon-based microfluidic chip and a detection method thereof. Background Art

[0002] Mycoplasma pneumoniae (MP) is a smallest prokaryotic microorganism that can survive independently, between bacteria and viruses. Unlike typical bacteria, mycoplasma pneumoniae has no cell wall and is insensitive to certain antibacterial drugs that target bacterial cell walls (such as β-lactams). Mycoplasma pneumoniae is a common pathogen of CAP. A global survey on the etiology of CAP showed that mycoplasma pneumoniae accounted for 12% of CAP. In the American IDSA's CAP diagnosis and treatment guidelines, mycoplasma pneumoniae ranks second only to Streptococcus pneumoniae. The European Respiratory Society and the Clinical Microbiology and Infection Group's guidelines for the diagnosis and treatment of lower respiratory tract infections in adults also confirmed that atypical pathogens plus viral infections account for about 30% of CAP. To this day, mycoplasma pneumoniae is still one of the more serious infectious diseases. Therefore, early, rapid and correct diagnosis is of great significance for the treatment and prevention of the spread of this disease.

[0003] Mycoplasma pneumoniae belongs to the class Mollicutes, family Mycoplasma, genus Mycoplasma. It is the smallest microorganism known to reproduce in a non-living culture medium, with a length of 1-2 μm and a width of 0.1-0.2 μm. The genome of mycoplasma is extremely small and has no cell wall. The commonly used methods for etiological diagnosis of MP are culture, serology and PCR. The latter two are the main methods used in clinical practice.

[0004] Traditional Mycoplasma pneumoniae detection methods are far from meeting modern detection requirements due to their shortcomings such as long detection cycle, complex procedures, and numerous required reagents. Pathogen nucleic acid detection technologies represented by polymerase chain reaction (PCR) technology have some problems in practical applications, such as the need for special instruments for ordinary polymerase chain reaction (PCR) technology, and the disadvantages of easy cross contamination and cumbersome operation process. Although the fluorescent real-time quantitative polymerase chain reaction (real time PCR) technology has better solved the problem of cross contamination and simplified the operation process, the detection time is long, so it is not suitable for on-site rapid detection. Immunological detection technology is fast, simple and low-cost, but requires high-quality and high-stability monoclonal antibodies, otherwise the accuracy is not enough, and it can only be an auxiliary detection method at present. Therefore, the timely use of the latest achievements in the development of biotechnology is of great significance to meet the continuous improvement of pathogenic microorganism detection requirements. Among them, the rapid detection technology of nucleic acid based on silicon-based microfluidic chips is a great progress in pathogen nucleic acid detection technology, and the established silicon-based microfluidic chip technology platform has many advantages.

[0005] Therefore, a Mycoplasma pneumoniae gene detection kit and detection method with more comprehensive detection effect, higher specificity and lower missed detection rate is needed to solve the above problems. Summary of the invention

[0006] The purpose of the present invention is to provide an ultra-rapid detection kit for Mycoplasma pneumoniae based on a silicon-based microfluidic chip, thereby solving the problems in the prior art of Mycoplasma pneumoniae detection methods that are prone to cross contamination, cumbersome operation process, long time consumption and high missed detection rate.

[0007] In order to solve the above problems, the present invention adopts the following technical solutions:

[0008] According to a first aspect of the present invention, a Mycoplasma pneumoniae gene detection kit based on a silicon-based microfluidic chip is provided, comprising a rapid hot-start DNA polymerase, a PCR reaction mixture, a positive control and a negative control, and also comprising the following primers designed by the PCR fluorescent probe method with the Mycoplasma pneumoniae repMp1 gene as the target gene: an upstream primer F, a downstream primer R, and a detection probe P, wherein the three primers are respectively:

[0009] Upstream primer F: 5′-CGGTGAATACGTTCTCGGGT-3′;

[0010] Downstream primer R: 5′-GTGCTATCCTTGACATGCGC-3′;

[0011] Detection probe P: 5'-TGTACACACCGCCCGTC-3'.

[0012] Preferably, the concentrations of the upstream primer F and the downstream primer R are both 0.4-0.8 μM, and the concentration of the detection probe P is 0.2-0.4 μM.

[0013] More preferably, the concentrations of the upstream primer F and the downstream primer R are both 0.4 μM, and the concentration of the detection probe P is 0.2 μM.

[0014] Preferably, the concentration of the fast hot-start DNA polymerase is greater than or equal to 0.25 U / μL.

[0015] More preferably, the concentration of the rapid hot-start DNA polymerase is 0.25 U / μL.

[0016] Preferably, the PCR reaction mixture contains: 1×PCR Buffer, 0.3125-0.625 mM dNTPs, 0.4-0.8 μM upstream primer F / downstream primer R, and 0.2-0.4 μM detection probe P.

[0017] More preferably, the PCR reaction mixture contains: 1×PCR Buffer, 0.625 mM dNTPs, 0.4 μM upstream primer F / downstream primer R, and 0.2 μM detection probe P.

[0018] The positive control substance is the plasmid of Mycoplasma pneumoniae; and the negative control substance is water.

[0019] According to a second aspect of the present invention, there is provided a Mycoplasma pneumoniae gene detection method of the Mycoplasma pneumoniae gene detection kit as described above, comprising the following steps:

[0020] 1) Use a pathogenic microorganism DNA / RNA extraction kit to extract DNA as a sample template;

[0021] 2) Add 0.2 volume parts of fast hot start DNA polymerase, 18.8 volume parts of PCR reaction mixture, and 5 volume parts of sample template DNA into the reaction container, wherein the sequences of upstream primer F, downstream primer R, and detection probe P are:

[0022] Upstream primer F: 5′-CGGTGAATACGTTCTCGGGT-3′;

[0023] Downstream primer R: 5′-GTGCTATCCTTGACATGCGC-3′;

[0024] Detection probe P: 5′-TGTACACACCGCCCGTC-3′;

[0025] 3) After the reaction is completed, samples with an S-shaped amplification curve and a Ct value within the reference interval are positive, otherwise they are negative.

[0026] Preferably, the reaction conditions in step 3) are 95°C for 50s; 95°C for 6s, 60°C for 10s, for 45 cycles.

[0027] It should be understood that the rapid nucleic acid detection technology based on silicon-based microfluidic chips detects fluorescent signals by fluorescent quantitative PCR on microfluidic chips, and completes the detection within ten minutes under the action of ultrafast amplification enzymes. This rapid nucleic acid detection technology based on silicon-based microfluidic chips overcomes the inherent shortcomings of traditional PCR, such as long detection time, easy contamination and high detection cost. In addition, this detection method has low technical requirements for detection personnel, and the actual operation is extremely simple. It does not require special reagents and instruments and equipment, which is conducive to the establishment of a low-cost rapid screening system. Comparing silicon-based microfluidic technology with PCR technology (including real-time fluorescent quantitative PCR technology), it can be found that this technology is equivalent to or better than PCR technology in methodological indicators such as sensitivity, specificity and detection range, and the detection time is greatly reduced, and the instrument and detection costs are low.

[0028] According to the present invention, a rapid nucleic acid detection technology based on a silicon-based microfluidic chip is provided, which uses a rapid hot-start DNA polymerase and three primers designed according to the target gene sequence (i.e., upstream primer F, downstream primer R, and detection probe P) to specifically identify a specific region on the target sequence and quickly complete the detection within ten minutes. The present invention further optimizes the concentrations of the upstream primer F, the downstream primer R, and the detection probe P. When the optimized primer concentration is used, the Mycoplasma pneumoniae gene detection kit has a high sensitivity, an S-type amplification curve, and a Ct value within the reference interval. In the ultrafast reagent system, the concentration of the components in the PCR reaction mixture and the concentration of the rapid hot-start DNA polymerase are both greater than those of the traditional PCR reagent components, so that the probability of molecular and ion collisions in the microfluidic chip is greatly increased, and amplification and detection are completed in a short time under the action of the ultrafast amplification enzyme.

[0029] Compared with the prior art, the present invention has the following beneficial effects: 1) the detection kit of the present invention can complete rapid detection of the repMp1 target gene on a microfluidic chip within ten minutes, while the detection time of other technical platforms is at least 2 hours; 2) the specific conserved region of the nucleic acid sequence of the Mycoplasma pneumoniae repMp1 gene is selected for primer design, and three primers are used to ensure the accuracy and specificity of the detection kit of the present invention in detecting the Mycoplasma pneumoniae gene; 3) the gene rapid diagnosis kit of the present invention can determine the presence or absence of the target substance according to whether there is an S-type amplification curve and the Ct value is within the reference interval, so it has high specificity; 4) the gene rapid diagnosis kit of the present invention is easy to identify, and the results can be analyzed by software.

[0030] In summary, according to the present invention, a Mycoplasma pneumoniae gene detection kit is provided, which has more comprehensive detection effect, high specificity, good sensitivity, low missed detection rate, and is convenient, simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The results of the sensitivity experiment of fluorescence quantitative PCR using the Mycoplasma pneumoniae microfluidic chip in Example 2, wherein A, B, and C are the results of the sensitivity experiment of fluorescence quantitative PCR on samples obtained by diluting the DNA extracted from Mycoplasma pneumoniae in 10-fold ratios. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0033] Example 1 Preparation of Mycoplasma pneumoniae gene detection kit

[0034] 1.1 Synthesize oligodeoxyribonucleic acid primers using a DNA synthesizer according to the following sequence:

[0035] Upstream primer F: 5'-CGGTGAATACGTTCTCGGGT-3' (SEQ ID NO. 1);

[0036] Downstream primer R: 5'-GTGCTATCCTTGACATGCGC-3' (SEQ ID NO. 2);

[0037] Probe P: 5'-TGTACACACCGCCCGTC-3' (SEQ ID NO. 3);

[0038] 1.2 Prepare DNA polymerase and reverse transcriptase: Mix the rapid hot start DNA polymerase and reverse transcriptase in a container;

[0039] 1.3 Prepare PCR reaction mixture: the reaction mixture contains 1×PCR Buffer, 0.625mM dNTPs, 0.4μM primer F / R, and 0.2μM probe P, and is placed in a container;

[0040] 1.4 Prepare positive control: Mycoplasma pneumoniae plasmid, place in container;

[0041] 1.5 Preparation of negative control: deionized water.

[0042] 1.6 Assemble the above four containers into a test kit and seal it.

[0043] The preparation process is briefly described as follows:

[0044] 1) After synthesizing and purifying the upstream primer F, downstream primer R, and probe P, dilute them with 1×TE, test the concentration, and perform sampling quality inspection;

[0045] 2) Aseptically packaging the liquid prepared in the above steps 2) to 3) and sampling for quality inspection;

[0046] 3) diluting and preparing the positive control, packaging, and sampling for quality inspection;

[0047] 4) Prepare, package, and sample the negative control for quality inspection;

[0048] 5) Assemble the kit.

[0049] Example 2 Application of Mycoplasma pneumoniae gene detection kit

[0050] 1 Materials and methods

[0051] 1.1 Materials

[0052] 1.1.1 Pathogens

[0053] The present invention uses 14 pathogen strains, which are mainly from the Chinese Center for Disease Control and Prevention, Shanghai Center for Disease Control and Prevention, and Pudong New Area Center for Disease Control and Prevention. See Table 1 for details.

[0054] Table 1 Strain names and sources

[0055]

[0056] 1.2 Sample processing

[0057] 1.2.1 Take 400 μL of throat swab sample and add it to lysis solution, proteinase K and magnetic beads for lysis and nucleic acid adsorption;

[0058] 1.2.2 After the magnetic beads adsorbed with nucleic acid are washed three times and eluted once, sample template DNA is obtained.

[0059] 1.3 Reaction process of silicon-based microfluidic chip nucleic acid rapid detection technology using the kit of Example 1

[0060] 1.4 Reagent preparation and reaction

[0061] 1.4.1 For the reaction on a 24 μL microfluidic chip, first prepare the reaction system in a 200 μL PCR reaction tube: 0.2 μL of rapid hot start DNA polymerase, 18.8 μL of PCR reaction mixture, and 5 μL of sample template DNA.

[0062] 1.4.2 The prepared reaction system reagents were reacted on the microfluidic chip under the reaction conditions of 95°C for 50s; 95°C for 6s, 60°C for 10s, and 45 cycles.

[0063] 1.5 Post-reaction treatment

[0064] After the reaction is completed, if there is an S-shaped amplification curve and the Ct value is within the reference interval, it is positive, otherwise it is negative.

[0065] 1.6 Specificity test

[0066] 1.6.1 The pathogen microfluidic chip technology platform was used for testing and 14 pathogen strains were amplified. Those with an S-shaped amplification curve and a Ct value within the reference range were positive, otherwise they were negative, verifying the specificity of the method.

[0067] 1.6.2 For mixed nucleic acid testing of several pathogens, take 5 μL of equal volume mixture of DNA of Mycoplasma pneumoniae, Chlamydia pneumoniae, Streptococcus pneumoniae, and Haemophilus influenzae for testing.

[0068] 1.7 Sensitivity test

[0069] The DNA extracted from Mycoplasma pneumoniae was diluted to E3, E4, and E5 in a 10-fold ratio, and then tested. The results are shown in Figure 1 .

[0070] 1.8 Repeatability test

[0071] The specificity test and sensitivity test were repeated twice respectively.

[0072] 2 Results

[0073] 2.1 Establishment of a silicon-based microfluidic chip detection method for Mycoplasma pneumoniae genes

[0074] 2.2 Specificity test The results of Mycoplasma pneumoniae test were positive, 13 strains of non-Mycoplasma pneumoniae were negative, and the DNA mixture of Mycoplasma pneumoniae, Chlamydia pneumoniae, Streptococcus pneumoniae, and Haemophilus influenzae were positive, as shown in Table 2. The results showed that the kit had high specificity.

[0075] Table 2 Results of specificity test using the method of the present invention

[0076] Test samples result Test samples result Negative Control Negative Haemophilus influenzae Negative Positive Control Positive Klebsiella pneumoniae Negative Ureaplasma urealyticum Negative Staphylococcus aureus Negative Mycoplasma genitalium Negative Mycobacterium tuberculosis Negative Mycoplasma hominis Negative Legionella pneumophila Negative Chlamydia pneumoniae Negative Pseudomonas aeruginosa Negative Streptococcus pneumoniae Negative Bordetella pertussis Negative Escherichia coli Negative Mycoplasma pneumoniae Positive

[0077] 2.3 Sensitivity test

[0078] The Mycoplasma pneumoniae DNA microfluidic chip method can detect a copy number of 1000 copies / mL. The microfluidic chip PCR results are shown in Figure 1 .

[0079] 2.4 Repeatability test The specificity test was repeated twice, and the results were consistent. The sensitivity test was repeated twice, and the magnitude was consistent.

[0080] Example 3 Screening of Primer and Probe Concentrations of Mycoplasma Pneumoniae Gene Detection Kit

[0081] 1 Materials and methods

[0082] 1.1 Materials

[0083] Plasmid.

[0084] 1.2 The reaction process of the silicon-based microfluidic chip nucleic acid rapid detection technology was carried out using the kit of Example 1.

[0085] 1.3 Screening of primer and probe concentration

[0086] Two sets of final concentrations were designed. The first set had a primer concentration of 0.2 μM and a probe concentration of 0.1 μM. The second set had a primer concentration of 0.4 μM and a probe concentration of 0.2 μM.

[0087] 1.4 Reagent preparation and reaction

[0088] 1.4.1 For the reaction on a 24 μL microfluidic chip, first prepare the reaction system in a 200 μL PCR reaction tube: 0.2 μL of rapid hot start DNA polymerase, 18.8 μL of PCR reaction mixture, and 5 μL of sample template DNA.

[0089] 1.4.2 The prepared reaction system reagents were reacted on the microfluidic chip under the reaction conditions of 95°C for 50s; 95°C for 6s, 60°C for 10s, and 45 cycles.

[0090] 2 Results

[0091] The results of the first set of primer-probe concentrations showed no starting line and no Ct value; the results of the second set of primer-probe concentrations showed an amplification curve and a Ct value.

[0092] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A Mycoplasma pneumoniae gene detection kit based on a silicon-based microfluidic chip, comprising a rapid hot-start DNA polymerase, a PCR reaction mixture, a positive control substance and a negative control substance, characterized in that: The method also includes the following primers designed by PCR fluorescent probe method with the Mycoplasma pneumoniae repMp1 gene as the target gene: an upstream primer F, a downstream primer R, and a detection probe P. The three primers are: Upstream primer F: 5′-CGGTGAATACGTTCTCGGGT-3′; Downstream primer R: 5′-GTGCTATCCTTGACATGCGC-3′; Detection probe P: 5'-TGTACACACCGCCCGTC-3'.

2. The Mycoplasma pneumoniae gene detection kit according to claim 1, characterized in that: The concentrations of the upstream primer F and the downstream primer R are both 0.4-0.8 μM, and the concentration of the detection probe P is 0.2-0.4 μM.

3. The Mycoplasma pneumoniae gene detection kit according to claim 2, characterized in that: The concentrations of the upstream primer F and the downstream primer R are both 0.4 μM, and the concentration of the detection probe P is 0.2 μM.

4. The Mycoplasma pneumoniae gene detection kit according to claim 1, characterized in that: The concentration of the rapid hot-start DNA polymerase is greater than or equal to 0.25 U / μL.

5. The Mycoplasma pneumoniae gene detection kit according to claim 4, characterized in that: The concentration of the rapid hot-start DNA polymerase is 0.25 U / μL.

6. The Mycoplasma pneumoniae gene detection kit according to claim 1, characterized in that: The PCR reaction mixture contains: 1×PCR Buffer, 0.3125-0.625 mM dNTPs, 0.4-0.8 μM upstream primer F / downstream primer R, and 0.2-0.4 μM detection probe P.

7. The Mycoplasma pneumoniae gene detection kit according to claim 6, characterized in that: The PCR reaction mixture contains: 1×PCR Buffer, 0.625 mM dNTPs, 0.4 μM upstream primer F / downstream primer R, and 0.2 μM detection probe P.

8. The Mycoplasma pneumoniae gene detection kit according to claim 1, characterized in that: The positive control substance is the plasmid of Mycoplasma pneumoniae; and the negative control substance is water.

9. A Mycoplasma pneumoniae gene detection method according to the Mycoplasma pneumoniae gene detection kit according to any one of claims 1 to 8, which is not used for diagnosis and treatment of diseases, characterized in that: The steps include: 1) Use a pathogenic microorganism DNA / RNA extraction kit to extract DNA as a sample template; 2) Add 0.2 volume parts of fast hot start DNA polymerase, 18.8 volume parts of PCR reaction mixture, and 5 volume parts of sample template DNA into the reaction container, wherein the sequences of upstream primer F, downstream primer R, and detection probe P are: Upstream primer F: 5′-CGGTGAATACGTTCTCGGGT-3′; Downstream primer R: 5′-GTGCTATCCTTGACATGCGC-3′; Detection probe P: 5′-TGTACACACCGCCCGTC-3′; 3) After the reaction is completed, samples with an S-shaped amplification curve and a Ct value within the reference interval are positive, otherwise they are negative.

10. The method for detecting Mycoplasma pneumoniae gene based on silicon-based microfluidic chip according to claim 9, characterized in that: The reaction conditions described in step 3) are 95°C for 50s; 95°C for 6s, 60°C for 10s, and 45 cycles.

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