Method for directly amplifying pathogens in throat swab sample

Abstract

The invention provides a method for directly amplifying pathogens in a throat swab sample, which comprises the following steps of: preparing a throat swab sample nucleic acid releasing agent from NP-40, Tris-base, EDTA (Ethylene Diamine Tetraacetic Acid), Chelex-100 and KCl, collecting the throat swab sample in normal saline during direct amplification, fully oscillating, taking 1mL of the throat swab sample, centrifuging 12,000 g of the throat swab sample for 10 minutes, discarding supernatant, retaining precipitate, adding 100 microliters of the throat swab sample nucleic acid releasing agent according to claim 1, shaking for 30-60 seconds by hand, and detecting the pathogens in the throat swab sample. And standing for 1-2 minutes to obtain a nucleic acid template for PCR (Polymerase Chain Reaction) detection. The method greatly simplifies the nucleic acid purification process of a clinical sample, greatly shortens the detection time, and has the advantages of low cost and convenience in use.

Description

Technical Field

[0001] The present invention relates to the technical field, and in particular to a method for directly amplifying pathogens in a throat swab sample. Background Art

[0002] There are many types of respiratory pathogens that can cause respiratory infections. The symptoms of respiratory infections caused by different pathogens are similar, but the treatment methods vary greatly. Therefore, rapid and accurate diagnosis of specific pathogens is the key basis for clinical formulation of specific treatment methods.

[0003] The conventional respiratory tract testing methods are mainly the following:

[0004] 1. Cultivation method

[0005] By collecting respiratory samples and inoculating them on a suitable culture medium, creating conditions suitable for the growth of microorganisms, and culturing them for a certain period of time, the growth of microorganisms is observed to determine whether specific microorganisms are present in the sample and their quantity. The advantages of the culture method are that the results are intuitive and reliable, and some common microorganisms can be identified more accurately. The disadvantages are that the operation is complicated, time-consuming, and cannot meet the needs of rapid testing. The detection range is limited, and only culturable microorganisms can be detected, and some difficult-to-culture microorganisms cannot be detected. In addition, it has high requirements for technical personnel and is limited in clinical use.

[0006] 2. Molecular biology detection methods

[0007] Molecular biology detection methods mainly include nucleic acid molecular hybridization, polymerase chain reaction (PCR), gene sequencing, gene chip and other technologies. These methods are based on the specificity and variability of nucleic acids, and diagnose human conditions and diseases by detecting the presence, defects or abnormal expression of genes.

[0008] 1) Nucleic acid molecule hybridization

[0009] Nucleic acid molecular hybridization technology is a process in which two nucleic acid single strands from different sources form double strands according to the principle of base complementary pairing under certain conditions. The main methods include fluorescence in situ hybridization (FISH), Southern blot hybridization, Northern blot hybridization, dot hybridization, etc. FISH technology is often used to detect chromosomal abnormalities, such as tumor detection and prenatal diagnosis. Southern blot hybridization and Northern blot hybridization are used to detect the presence of DNA and RNA, respectively.

[0010] 2) Polymerase chain reaction (PCR)

[0011] PCR is a technique for amplifying specific DNA fragments in vitro. Primers bind to specific DNA fragments and synthesize new DNA chains under the action of DNA polymerase. PCR technology is widely used in the detection and identification of infectious pathogens such as bacteria, viruses, and parasites. It is fast and sensitive.

[0012] 3) Gene sequencing

[0013] Gene sequencing is the process of determining the sequence of specific DNA fragments to understand the genetic composition and characteristics. Gene sequencing can be used to identify pathogen species, understand the structure and variation of pathogen genomes, and provide a basis for formulating treatment plans.

[0014] 4) Gene chip

[0015] Gene chip is a technology that fixes a large number of biological probes on a solid matrix, forming detectable signals through specific binding, and realizing high-throughput detection of pathogens. Gene chip has the advantages of high efficiency, rapidity and high sensitivity, and is suitable for the detection and identification of a variety of infectious pathogens.

[0016] 3. Immunization

[0017] The main immunological methods include immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence assay, etc., which are mainly used for antigen or antibody detection. The immunological method has mature technology, low cost, and is widely used in clinical practice. However, it has low sensitivity and is gradually being replaced by molecular biology detection methods in some fields.

[0018] These methods have the advantages of being fast and accurate in disease diagnosis and are widely used in the prevention, diagnosis and treatment of clinical diseases, providing data support for personalized medicine.

[0019] Molecular biology methods are highly sensitive and specific, and have become one of the most important means of detecting respiratory pathogens. However, molecular biology methods have high requirements for sample nucleic acid purity, and require nucleic acid purification reagents with good performance to play the advantages of detection. Nucleic acid purification methods are cumbersome and time-consuming. Although there are automatic extractors that can solve most of the manual operations, sample pretreatment still troubles a large number of clinical laboratory personnel. Therefore, it is urgent to develop a PCR method for direct amplification of pathogens in throat swab samples. Summary of the invention

[0020] The traditional nucleic acid purification process is complicated, time-consuming and has poor repeatability. In order to overcome the shortcomings in the prior art, the present invention provides a method for directly amplifying pathogens in throat swab samples, which utilizes a throat swab sample releaser to fully release pathogen nucleic acids, and directly amplifies the throat swab samples with the help of a high-sensitivity PCR system to facilitate rapid diagnosis of pathogens.

[0021] To achieve the above object, the present invention provides a throat swab sample nucleic acid release agent, which comprises the following components, wherein per unit of the throat swab sample nucleic acid release agent,

[0022] NP-40 0.5-1.0 volume parts Tris-base 100-125 parts by mass EDTA 30.0-40.0 parts by mass Chelex-100 90.0-100.0 parts by mass KCl 1.0-1.5 parts by mass

[0023] The unit of mass parts is gram or its proportional mass unit, and the unit of volume parts is milliliter or its proportional volume unit.

[0024] Preferably, the pH of the throat swab sample nucleic acid releaser is 7.8-8.2.

[0025] Preferably, water is also included.

[0026] The present invention provides a method for preparing the above-mentioned throat swab sample nucleic acid releaser, which is: take a corresponding number of the substances in claim 1, measure 500-800 mL of purified water, add the above component reagents, stir thoroughly until completely dissolved, adjust the pH value to 7.8-8.0 with dilute hydrochloric acid, and make the volume to 1000 mL with purified water.

[0027] The present invention provides a method for directly amplifying pathogens in a throat swab sample, which comprises: using the throat swab sample nucleic acid releaser to directly amplify Mycoplasma pneumoniae.

[0028] Preferably, the throat swab sample is collected in physiological saline, 1 mL is taken after sufficient shaking, centrifuged at 12000g for 10 min, the supernatant is discarded, the precipitate is retained, 100 μL of the throat swab sample nucleic acid releaser described in claim 1 is added, and it is shaken by hand for 30-60 s and allowed to stand for 1-2 min. It can be used as a nucleic acid template for PCR detection.

[0029] Preferably, the PCR reaction solution is,

[0030]

[0031]

[0032] Preferably, the PCR reaction procedure is,

[0033]

[0034] The beneficial effects of the present invention are as follows:

[0035] The nucleic acid releaser provided by the present invention does not require nucleic acid purification of clinical samples. The releaser prepared by a special formula is combined with a corresponding high-sensitivity PCR reaction system. Different from traditional PCR detection, PCR amplification can be directly performed, which greatly simplifies the nucleic acid purification process of clinical samples and greatly shortens the detection time. It has the advantages of low cost and easy use, provides assistance for rapid identification of pathogens, is conducive to quickly guiding clinicians to formulate treatment plans, and shortens the patient's disease course. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the PCR amplification curve of the present invention. DETAILED DESCRIPTION

[0037] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present application will be further described below in conjunction with specific embodiments.

[0038] Terminology explanation:

[0039] Fluorescence quantitative PCR: A method that uses fluorescent chemicals to measure the total amount of products after each polymerase chain reaction (PCR) cycle in a nucleic acid amplification reaction. A method that uses internal or external reference methods to quantitatively analyze specific nucleic acid sequences in the sample to be tested.

[0040] Primer: Two artificially synthesized oligonucleotide sequences, one primer is complementary to a DNA template strand at one end of the target gene, and the other primer is complementary to the other DNA template strand at the other end of the target gene.

[0041] Probe: A nucleic acid sequence with a known sequence and a detectable label that is complementary to the target gene.

[0042] Throat swab: A disposable medical swab is used to collect oral or throat secretions for testing for respiratory pathogens.

[0043] NP-40: A nonionic surfactant mainly composed of a longer hydrophilic polyoxyethylene chain and a shorter hydrophobic octyl chain.

[0044] BSA: bovine serum albumin.

[0045] DMSO: Dimethyl sulfoxide, a sulfur-containing organic compound with the molecular formula (CH3)2SO, is a colorless, odorless, transparent liquid at room temperature. It is an important organic solvent that can reduce nonspecific reactions in PCR amplification.

[0046] Embodiment 1:

[0047] 1. According to the characteristics of throat swab samples, a throat swab sample nucleic acid releaser is prepared, and its formula is as follows:

[0048] Recipe 1:

[0049] Reagent name Mass / Volume NP-40 1mL Tris-base 121g EDTA 37.2g Chelex-100 100g KCl 1.5g pH 7.8-8.2 Water to volume 1000mL

[0050] Recipe 2:

[0051]

[0052]

[0053] Recipe 3:

[0054] Reagent name Mass / Volume Tris-base 121g EDTA 37.2g Chelex-100 100g KCl 1.5g pH 7.8-8.2 Water to volume 1000mL

[0055] Take 500-800mL of purified water, add the above reagents, stir thoroughly until completely dissolved, adjust the pH value to 7.8-8.0 with dilute hydrochloric acid, and make up to 1000mL with purified water.

[0056] 2. The releaser is used to process clinical throat swab samples, followed by direct PCR amplification testing.

[0057] 3. Take Mycoplasma pneumoniae resistance detection reagent as an example:

[0058] 3.1 Introduction to Mycoplasma pneumoniae

[0059] Mycoplasma pneumoniae can cause mycoplasma pneumonia, upper respiratory tract infection, bronchitis, lung abscess, immune hemolytic anemia, meningoencephalitis, myocarditis, pericarditis, nephritis, community-acquired pneumonia and other diseases. Macrolides are the first choice for the treatment of Mycoplasma pneumoniae infection. Clinically, macrolide-resistant strains have been isolated. The duration of fever, hospitalization time and duration of fever after medication in patients infected with resistant strains of Mycoplasma pneumoniae are significantly longer than those in patients infected with sensitive strains. The binding site of macrolide drugs is located in the V domain of 23S rRNA. The nucleotide point mutations in this region will reduce the affinity between antibiotics and ribosomes, thereby making Mycoplasma pneumoniae resistant. The mutation sites that have been found so far include 2063, 2064, 2067 and 2617. Among them, A2063G showed resistance to 14-ring macrolide antibiotics, A2064G showed resistance to 14- and 16-ring macrolide antibiotics, C2617G showed resistance to 14- and 15-ring macrolide antibiotics, and A2067G showed resistance to josamycin. Currently, only point mutations at sites 2063 and 2064 have been found in China, and the most common mutation is the mutation from A to G at site 2063. According to the restriction fragment length polymorphism analysis of the P1 gene, Mycoplasma pneumoniae is divided into type III, of which type I is further subdivided into 5 genetic subtypes (1a-1e) and type II is further divided into 3 subtypes (2a-2c).

[0060] 3.2 Detection of Mycoplasma pneumoniae-positive pharyngeal swab clinical samples

[0061] The Mycoplasma pneumoniae nucleic acid and drug resistance mutation site detection kit (fluorescence PCR method) (Jiangsu Mole Biotechnology Co., Ltd., registration certificate number: National Medical Device Registration No. 20143402242) was used to detect 10 clinical samples of Mycoplasma pneumoniae-positive throat swabs. The throat swab samples were collected in physiological saline, and after sufficient shaking, 1 mL was taken and respectively used for magnetic bead method nucleic acid purification reagent (Jiangsu Mole Biotechnology Co., Ltd. Registration Certificate No.: Sutai Medical Device Preparation 20200507) (nucleic acid purification steps refer to the reagent instructions), and another 1 mL was taken and centrifuged at 12000g for 10 minutes, the supernatant was discarded, the precipitate was retained, 100 μL of the nucleic acid release agent of the present invention was added, and it was shaken by hand for 30-60 seconds and allowed to stand for 1-2 minutes, and then it can be used as a nucleic acid template for PCR detection.

[0062] The samples extracted with magnetic beads and treated with release agents were tested in parallel. Two wells of each sample were tested repeatedly, and the results of the sample tests were analyzed.

[0063] 3.3PCR detection:

[0064] 3.3.1 Preparation of PCR reaction solution:

[0065]

[0066]

[0067] 3.3.2PCR reaction procedure:

[0068]

[0069] The collection of fluorescence signals was set to FAM (resistance to Mycoplasma pneumoniae) and VIC (Mycoplasma pneumoniae), and the data collection was set to 64°C. When using the ABI7500 instrument, please select "none" in the "Quencher" column and the "Passive reference" column.

[0070] 3.4 PCR amplification curve Figure 1 .

[0071] The blue amplification curve is the drug-resistant mutation of Mycoplasma pneumoniae, labeled with FAM fluorescence, and the green amplification curve is Mycoplasma pneumoniae, labeled with VIC fluorescence.

[0072] 3.5 PCR test results

[0073] 3.5.1 Test results of formula 1:

[0074]

[0075] 3.5.2 Test results of formula 2:

[0076]

[0077]

[0078] 3.5.3 Test results of formula 3:

[0079]

[0080] It can be seen from the test data of the above 10 clinical samples of Mycoplasma pneumoniae that the direct PCR amplification effect of the samples treated with the release agent of Formula 1 is equivalent to that of the samples extracted with magnetic beads, and the direct amplification effects of samples 5, sample 6, sample 7, sample 8 and sample 10 are even better, while the Ct values ​​of the direct amplification results of the release agents of Formula 2 and Formula 3 are pushed back to varying degrees. Samples 5, 6, 7, 8, and 10 of the release agent of Formula 2 have no amplification signals, and samples 3 and sample 9 in the release agent of Formula 3 have amplification signal fluctuations, and samples 8 and sample 10 have no amplification signals, indicating that the release agent effects of these two formulas are worse than the PCR amplification effect of magnetic bead-extracted nucleic acid. It can be seen that the direct amplification method of the release agent of Formula 1 of the present invention can replace the traditional PCR detection after nucleic acid purification.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A throat swab sample nucleic acid release agent, characterized in that: The following components are included in each unit of the throat swab sample nucleic acid release agent: The unit of mass part is gram or its proportional mass unit, the unit of volume part is milliliter or its proportional volume unit, and the unit of mass-to-volume ratio is g / ml.

2. The pharyngeal swab sample nucleic acid releaser according to claim 1, characterized in that: The pH of the throat swab sample nucleic acid releaser is 7.8-8.

2.

3. The pharyngeal swab sample nucleic acid releaser according to claim 1, characterized in that: Also includes water.

4. The method for preparing the throat swab sample nucleic acid release agent according to claim 1, characterized in that: Take the corresponding amount of the substance in claim 1, measure 500-800 mL of purified water, add the above components and reagents, stir until completely dissolved, adjust the pH value to 7.8-8.0 with dilute hydrochloric acid, and make up to 1000 mL with purified water.

5. A method for direct amplification of pathogens in throat swab samples, characterized in that: The pharyngeal swab sample nucleic acid releaser according to claim 1 is used to directly amplify Mycoplasma pneumoniae.

6. The method for direct amplification of pathogens in throat swab samples according to claim 5, characterized in that: The throat swab sample is collected in physiological saline, and 1 mL is taken after sufficient shaking, centrifuged at 12000g for 10 min, the supernatant is discarded, and the precipitate is retained. 100 μL of the throat swab sample nucleic acid releaser described in claim 1 is added, and it is shaken by hand for 30-60 s and allowed to stand for 1-2 min. It can be used as a nucleic acid template for PCR detection.

7. The method for direct amplification of pathogens in throat swab samples according to claim 6, characterized in that: The PCR reaction solution is:

8. The method for direct amplification of pathogens in throat swab samples according to claim 6, characterized in that: The PCR reaction program is:

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