CN114480689A

CN114480689A - RPA method for detecting streptococcus pneumoniae, special primer and probe thereof and application

Info

Publication number: CN114480689A
Application number: CN202210164196.2A
Authority: CN
Inventors: 龚正华; 唐敏; 夏震遥; 卢甜
Original assignee: Shenzhen Yanyuan Biotechnology Co ltd
Current assignee: Shenzhen Yanyuan Biotechnology Co ltd
Priority date: 2022-02-22
Filing date: 2022-02-22
Publication date: 2022-05-13

Abstract

The invention discloses an RPA method for detecting streptococcus pneumoniae, a special primer, a probe and application thereof, wherein the special primer and the probe are designed based on a streptococcus pneumoniae specific conserved sequence and have oligonucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4. The invention applies the novel constant temperature amplification technology RPA to the detection of streptococcus pneumoniae for the first time, the method simulates the enzyme reaction process of DNA replication in vivo, and the amplification of a DNA template is carried out by depending on specific enzyme and protein combination, including recombinase, single-chain binding protein and DNA polymerase, the amplification of a specific nucleic acid sequence can be realized at the constant temperature of 37 ℃, and the visual discrimination of the amplification product can be realized by a lateral chromatography nucleic acid test paper strip. The detection method established by the invention has the advantages of sensitivity reaching 10 copy number/mu L, high specificity and low requirement on hardware equipment, can finish detection within 30min, does not need to carry out complex treatment on samples, is suitable for field detection, and is suitable for popularization and application.

Description

RPA method for detecting streptococcus pneumoniae, special primer and probe thereof and application

Technical Field

The invention belongs to the technical field of biology, relates to the molecular biology of streptococcus pneumoniae, relates to a method for detecting streptococcus pneumoniae and application thereof, and particularly relates to a method for quickly detecting streptococcus pneumoniae by utilizing recombinase polymerase isothermal amplification technology (RPA technology), a special primer and a probe thereof and application thereof.

Background

Fastidious bacteria are the most common pathogenic bacteria of community infection, can cause severe invasive diseases such as bacteremia, meningitis, pneumonia and the like, and are particularly serious in people with low immunity such as children and the like. Early and rational use of antibacterial drugs in the early stages of infection is very important for both treatment and prognosis. However, the isolation and identification of fastidious bacteria takes a long time, has strict requirements on the culture environment and is very easy to be limited by laboratory, personnel and technical differences, thereby seriously affecting the diagnosis of fastidious bacteria infected persons and delaying the treatment time of clinical patients. And streptococcus pneumoniae is the largest group among them. The patent provides a streptococcus pneumoniae inspection method based on an RPA technology, which is used for quickly, efficiently and accurately inspecting streptococcus pneumoniae; the method is beneficial to judging the state of the streptococcus pneumoniae infection and guiding the medication of antibacterial drugs; and can effectively shorten the diagnosis and detection time, save the detection cost, and have great clinical significance and social and economic values.

Fastidious bacteria are bacteria which have strict nutrient requirements, are difficult to grow or do not grow in a common culture medium, and can slowly grow by adding special factors or other nutrient components in the in-vitro culture process, and mainly comprise streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis, neisseria meningitidis, legionella, bordetella and the like. The most of the fastidious bacteria which are common in clinic are normal flora or inhabitation bacteria of human respiratory tract, which do not cause diseases when the human immunity is normal, but can cause serious respiratory system inflammation or enter the circulatory system to cause infection of other parts under the condition of insufficient organism immunity, such as children patients or immunosuppression. Research shows that fastidious bacteria are important pathogenic bacteria in children infectious diseases, and can cause respiratory tract infection, otitis media, septicemia, meningitis and the like. In children patients, the detection rate and the drug resistance rate of fastidious bacteria are obviously higher than those of adults. And among the fastidious bacteria positive specimens, the respiratory tract infection specimens account for 90 percent. Therefore, in clinical examination of patients, attention should be paid to the detection of fastidious bacteria in respiratory tract of children patients.

Respiratory tract infection can be mainly divided into two types, namely upper respiratory tract infection and lower respiratory tract infection, viruses and bacteria are main pathogens of the upper respiratory tract infection, and fungi, mycoplasma, viruses and bacteria are main factors causing the lower respiratory tract infection. Aiming at diagnosis and treatment of the diseases, the primary condition is accurate judgment of pathogenic bacteria in clinical examination, so that the medication rationality is improved, and a certain delay effect on the development of drug resistance is realized. In current clinical tests, the isolation and identification of respiratory pathogens remains the gold standard for the diagnosis of infected patients. However, the fastidious bacteria have extremely high requirements on factors such as nutrition and environment in the culture process, and because various laboratories have differences in environment, personnel configuration and technology, the conventional bacterial culture method is easy to miss diagnosis, so that the overall detection rate of the fastidious bacteria is only 20% -60%. Clinically, infection caused by fastidious bacteria is difficult to diagnose and treat accurately, so that the unreasonable use of antibacterial drugs is caused, and the optimal treatment time of patients is delayed. For example, severe lung infection is taken as an example, proper antibacterial drugs can be selected for determining pathogenic bacteria, so that the risk of drug resistance is reduced. In clinical practice at present, the important value of etiology diagnosis is not paid enough attention, empirical diagnosis is mainly used, etiology fuzzy diagnosis such as unknown pneumonia and chronic pneumonia is common, and the phenomenon of microorganism detection capability and clinical requirement disjointing is common. In the case of streptococcus pneumoniae, the typical forms are uplift, moist, umbilicate-shaped, grass-green hemolysis and difficult to distinguish from other bacteria in streptococcus; the haemophilus influenzae is in the form of drop-shaped microcolonies, insoluble in blood, colorless and transparent, and similar to neisseria. However, it is reported that when different sources of agar plates are used for the isolation and identification of fastidious bacteria, the bacteria may have different morphologies due to slight differences in the nutrient components contained therein, which increases the difficulty of identification. In addition, the related scholars indicate that the detection rate of fastidious bacteria such as streptococcus pneumoniae and haemophilus influenzae is obviously reduced along with the prolonging of the sample sending time, and the samples for the detection of fastidious bacteria are also not suitable for refrigeration, and the timeliness requirement of the sample sending is extremely high because the refrigerated samples easily cause the death of the fastidious bacteria. Therefore, how to detect fastidious bacteria infection quickly, effectively and accurately in clinic is a major challenge to clinical laboratories.

In recent years, isothermal nucleic acid Amplification technology has been rapidly developed, wherein Recombinase Polymerase isothermal Amplification (RPA) developed by british TwistDx Inc is known as a nucleic acid detection technology capable of replacing PCR, which is based on Recombinase Polymerase-mediated Amplification principle, simulates an enzymatic reaction process of in vivo DNA replication, amplifies a DNA template depending on specific enzyme and protein combination (Recombinase, single-strand binding protein and DNA Polymerase), can realize Amplification of a specific nucleic acid sequence at a constant temperature of 25-43 ℃, and an Amplification product can realize visual discrimination through a lateral chromatography detection test strip. The technology has low requirement on hardware equipment and short reaction time, does not need to carry out complex treatment on samples, and is particularly suitable for the fields of in-vitro diagnosis, food safety, biological safety and the like.

Disclosure of Invention

In view of the above, the present invention aims to provide an RPA method for detecting streptococcus pneumoniae, a primer and a probe dedicated for the same, and uses thereof, and a recombinase polymerase isothermal amplification technique (RPA technique) can be used to quickly, simply and specifically detect streptococcus pneumoniae.

The adopted technical scheme is as follows:

the invention relates to a non-diagnostic method for detecting streptococcus pneumoniae based on RPA, which comprises the following steps:

s1, taking genome DNA of a sample to be detected as a template, and carrying out an RPA reaction under the marks of a primer group and a probe; the primer group and the probe are designed according to a streptococcus pneumoniae specific conserved sequence, the streptococcus pneumoniae specific conserved sequence has a nucleotide sequence shown in SEQ ID No.1, a forward primer in the primer group has an oligonucleotide sequence shown in SEQ ID No.2, a reverse primer has an oligonucleotide sequence shown in SEQ ID No.3, and the 5' end is marked with biotin; the probe has an oligonucleotide sequence shown in SEQ ID NO.4, fluorescein is marked at the 5 'end, an extension blocking group is added at the 3' end, and tetrahydrofuran is added between 34 th and 35 th bases;

s2, detecting the recovered RPA product by using the lateral chromatography nucleic acid detection test strip, wherein a detection line and a quality control line are both displayed, and a positive result is judged; the detection line is not shown, the quality control line is shown, and the judgment result is a negative result; the quality control line is not shown, and whether the detection line is shown or not is judged that the result is invalid.

Further, the fluorescein is carboxyfluorescein FAM or FITC fluorescein, and the extension blocking group is a phosphate group.

Furthermore, the length of the probe is 49bp, wherein the 5 'end is 34bp, and the 3' end is 15 bp.

Further, in S1, a primer group and a probe are used in a 50 μ L RPA reaction system to perform an RPA reaction, wherein the concentration of the forward primer is 10 μmol/L, the concentration of the reverse primer is 5 μmol/L, the concentration of the probe is 10 μmol/L, the concentration of magnesium ions is 280 μmol/L, and the sample adding amount of the template is 1 μ L; a premix of 2.1. mu.L of the forward primer, 2.1. mu.L of the reverse primer, 0.6. mu.L of the probe, 1. mu.L of the sample, 12.2. mu.L of DNase-free and RNase-free water and 29.5. mu.L of buffer was added to a 0.2mL TwistAmp nfo reaction tube containing lyophilized enzyme powder, and then 2.5. mu.L of a magnesium acetate solution was added to the cap of the reaction tube; throwing the magnesium acetate solution on the cover of the reaction tube, fully and uniformly mixing, amplifying at 37 ℃ for 20min, taking out the reaction tube for fully and uniformly mixing in the 4 th min of reaction, and then putting the reaction tube back to the reaction device for continuous amplification; in S2, the sample adding amount is 5 μ L during detection, and the color development time of the test strip is controlled within 3-5 min.

The kit for detecting the streptococcus pneumoniae comprises a primer group and a probe; the primer group and the probe are designed according to a streptococcus pneumoniae specific conserved sequence, the streptococcus pneumoniae specific conserved sequence has a nucleotide sequence shown in SEQ ID No.1, a forward primer in the primer group has an oligonucleotide sequence shown in SEQ ID No.2, a reverse primer has an oligonucleotide sequence shown in SEQ ID No.3, and the 5' end is marked with biotin; the probe has an oligonucleotide sequence shown in SEQ ID NO.4, fluorescein is marked at the 5 'end, an extension blocking group is added at the 3' end, and tetrahydrofuran is added between 34 th and 35 th bases.

The invention relates to an RPA special primer for detecting streptococcus pneumoniae, which is designed according to a streptococcus pneumoniae specific conserved sequence, wherein the streptococcus pneumoniae specific conserved sequence has a nucleotide sequence shown in SEQ ID No. 1; the forward primer in the primers has an oligonucleotide sequence shown in SEQ ID NO.2, the reverse primer has an oligonucleotide sequence shown in SEQ ID NO.3, and the 5' end is marked with biotin.

The RPA probe for detecting the streptococcus pneumoniae is designed according to a streptococcus pneumoniae specific conserved sequence, and the streptococcus pneumoniae specific conserved sequence has a nucleotide sequence shown in SEQ ID No. 1; the probe has an oligonucleotide sequence shown in SEQ ID NO.4, fluorescein is marked at the 5 'end, an extension blocking group is added at the 3' end, and tetrahydrofuran is added between 34 th and 35 th bases.

The above-mentioned kit of the invention is used for the non-diagnostic use for the detection of streptococcus pneumoniae.

The RPA special primer is used for the non-diagnostic purpose of detecting streptococcus pneumoniae.

The RPA probe provided by the invention is used for non-diagnostic purposes for detecting streptococcus pneumoniae.

In the above, the first object of the present invention is to provide primers for detecting Streptococcus pneumoniae, including two forward primers and two reverse primers. The primers are designed according to the conserved gene of the streptococcus pneumoniae, and simultaneously, the homologous sequence of the gene is contrastively analyzed through software, so that the conserved region of the streptococcus pneumoniae gene is further determined, and the region contains a nucleotide fragment of 190 bases and has a nucleotide sequence shown in SEQ ID NO. 1. Specific primers are screened and designed from the SEQ ID NO.1 sequence. The screened specific primers comprise a forward primer PrF1 and a reverse primer PrR2, the two primers are provided with oligonucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO.3, the length of the primers is more than 30bp, and the 5' end of the reverse primer is marked with Biotin (Biotin). The double-stranded DNA obtained after amplification of the forward primer and the reverse primer is labeled with biotin.

It is a second object of the present invention to provide a probe for detecting Streptococcus pneumoniae. The probe is designed according to a specific conserved sequence of streptococcus pneumoniae, and a conserved region of the genes of the children respiratory tract fastidious bacteria is further determined by comparing and analyzing gene homologous sequences through software, wherein the region contains a nucleotide fragment of 190 bases and has a nucleotide sequence shown in SEQ ID NO. 1. Screening and designing a specific probe from the sequence of SEQ ID NO.1, wherein the designed probe has an oligonucleotide sequence shown in SEQ ID NO.4, the 5 'end of the sequence is marked with fluorescein FAM, the 3' end of the sequence is added with an extension blocking group (such as a phosphate group), and Tetrahydrofuran (THF) is inserted between 34 th and 35 th bases.

The length of the probe is 49bp, wherein the 5 'end is 34bp, and the 3' end is 15 bp.

The probe consists of fluorescein (carboxyfluorescein FAM), a 5 ' terminal sequence, Tetrahydrofuran (THF), a 3 ' terminal sequence and a 3 ' terminal extension blocking group (phosphate group).

The probe is annealed with amplified DNA marked with Biotin, and nfo enzyme in an RPA system cuts off the probe at a THF position, so that the probe can continue to extend at a 3' end under the action of polymerase, and finally an amplification product marked by FAM and Biotin is obtained.

The third purpose of the invention is to provide an RPA detection method for rapidly detecting streptococcus pneumoniae, which adopts the RPA primer and the probe to carry out amplification and combines a lateral chromatography nucleic acid detection test strip (hybrid 2T, Milenia Biotec GmbH, Germany) to carry out visual judgment.

The invention relates to a method for detecting RPA of streptococcus pneumoniae, which comprises the following steps:

(1) taking the genome DNA of a sample to be detected as a template, and carrying out RPA reaction under the marks of the primer group and the probe;

(2) and (5) judging a result: detecting the recovered RPA product by using the lateral chromatography nucleic acid detection test strip, wherein a detection line and a quality control line are both displayed, and a judgment result is a positive result; the detection line is not shown, the quality control line is shown, and the judgment result is a negative result; the quality control line is not shown, and whether the detection line is shown or not is judged that the result is invalid.

Preferably, the method for detecting the RPA of the streptococcus pneumoniae comprises the following specific steps:

(1) amplification reagent preparation and loading: mu.l of forward primer 2.1. mu.l, 5. mu. mol/L of reverse primer 2.1. mu.l, 10. mu. mol/L of probe 0.6. mu.l, 1. mu.l of sample, 12.2. mu.l of DNase-free and RNase-free water and 29.5. mu.l of buffer were combined to make a premix, which was added to a 0.2mL TwistAmpnfo reaction tube containing lyophilized enzyme powder. Then 2.5. mu.L of a 280mM magnesium acetate solution was added to the cap of the reaction tube.

(2) Amplification: throwing the magnesium acetate solution on the cover of the reaction tube, fully and uniformly mixing, amplifying at 37 ℃ for 20min, taking out the reaction tube in the 4 th min of reaction, fully and uniformly mixing, and then putting the reaction tube back to the reaction device for continuous amplification.

(3) And (5) judging a result: diluting 5 μ L of RPA amplification product to 100 μ L with PBST, detecting the recovered RPA product with the colloidal gold transverse flow test strip, and determining that the detection line and quality control line show positive result; the detection line is not shown, the quality control line is shown, and the judgment result is a negative result; the quality control line is not shown, and whether the detection line is shown or not is judged that the result is invalid.

In conclusion, the beneficial effects of the invention are as follows:

in a first aspect, the present invention provides a method for detecting an RPA of streptococcus pneumoniae, which adopts an RPA technique to detect a specific conserved target sequence of streptococcus pneumoniae, i.e., a conserved sequence of streptococcus pneumoniae, which can be used as one of marker genes of streptococcus pneumoniae.

In a second aspect, the RPA method for detecting streptococcus pneumoniae provided by the invention saves the detection time of streptococcus pneumoniae, can complete amplification within 20min at 37 ℃, can complete the whole detection process within 1 hour, and greatly shortens the detection time compared with the conventional PCR and the real-time fluorescent quantitative PCR which need several hours.

In the third aspect, the RPA method for detecting streptococcus pneumoniae provided by the invention reduces the reaction temperature, the RPA can complete the experiment only by keeping the temperature at 37 ℃, and the temperature is far lower than 60-95 ℃ of fluorescent quantitative PCR and 63 ℃ of LAMP.

In a fourth aspect, the RPA method for detecting streptococcus pneumoniae provided by the invention is simpler and more portable: the enzyme and other necessary matters required by amplification can be stored in a freeze-drying way, the enzyme and other necessary matters can be placed for a long time at normal temperature, only hydrolysis buffer solution, primers, probes and templates need to be added during amplification, magnesium ions are added for initiating reaction, and the sample does not need to be subjected to complex reaction.

In a sixth aspect, the RPA method for detecting streptococcus pneumoniae provided by the invention has high sensitivity and strong specificity. Can be used for on-site or bedside detection and has wide application prospect.

In summary, the invention applies the novel isothermal amplification technology RPA to the detection of streptococcus pneumoniae for the first time, the method simulates the enzyme reaction process of DNA replication in vivo, and amplifies a DNA template depending on specific enzyme and protein combination, including recombinase, single-strand binding protein and DNA polymerase, so that the amplification of a specific nucleic acid sequence can be realized at the constant temperature of 37 ℃, and the visual discrimination of the amplification product can be realized through a lateral chromatography nucleic acid test paper strip. The detection method established by the invention has the advantages of sensitivity reaching 10 copy number/mu L, high specificity and low requirement on hardware equipment, can finish detection within 30min, does not need to carry out complex treatment on samples, is suitable for field detection, and is suitable for popularization and application.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings related to the embodiments will be briefly described below.

FIG. 1 shows the combination of primers and probes for determining the detection method for detecting RPA in Streptococcus pneumoniae, E shows the determined primers, and the test results are shown in FIG. 1.

FIG. 2 shows the specificity of the detection method for detecting RPA of Streptococcus pneumoniae by using different genomic DNA templates for the test, wherein A is Streptococcus pyogenes, B is Streptococcus pneumoniae and C is Streptococcus agalactiae, and the test results are shown in FIG. 2.

Detailed Description

The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in the molecular cloning instructions, published by Sambrook et al, or according to the manufacturer's recommendations.

The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.

The RPA primer and the probe are synthesized by Shanghai Biotechnology company, and the qPCR primer and the probe are synthesized by Shanghai Biotechnology company, and all sequence determination work is completed by the Shanghai Biotechnology company.

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 illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1 design and screening of primers and probes for Streptococcus pneumoniae

(1) Design of primers and probes

The inventors have analytically determined, by file search, that the present invention uses a specific sequence of streptococcus pneumoniae as a target gene. A known template gene sequence, namely a nucleotide sequence shown in SEQ ID No.1 (or shown in Table 2), is obtained from an NCBI database, and the sequence is synthesized by Shanghai Biotechnology Limited company and used as a positive plasmid and used as a template in the processes of subsequent primer probe screening, reaction system optimization and the like. According to the principle of RPA primer and probe design, 2 primers and 1 probe were designed as shown in Table 1.

TABLE 1 sequences, primers and probes

TABLE 2 template Gene sequences

(2) Primer screening

Artificially synthesizing a sequence shown in SEQ ID NO.1 containing streptococcus pneumoniae, taking the plasmid as a template, and comprehensively combining a primer and a probe. And respectively carrying out RPA amplification on the primer combinations at 37 ℃, and screening out the primer probe combination with the highest amplification efficiency at 37 ℃ by taking the condition shown by the detection line of the lateral chromatography nucleic acid detection test strip as an index for evaluation and application of subsequent RPA detection.

A50. mu.L RPA reaction was selected as follows: 2.1. mu.L of forward primer 10. mu. mol/L, 2.1. mu.L of reverse primer 5. mu. mol/L, 0.6. mu.L of probe 10. mu. mol/L, 5.3X 10¹⁰1. mu.L copies/. mu.L template, 12.2. mu.L DNase-free and RNase-free water and 29.5. mu.L buffer, and 0.2mL of a solution containing the lyophilized enzyme powder

nfo reaction tube. Then 2.5. mu.L of 280mM magnesium acetate solution was added to the cap of the reaction tube, and in view of the high sensitivity of the RPA reaction and the susceptibility to false positive, the inventors set up a set of negative controls for each set of primer probe combination, the negative controls were not added with template, and the volume of the template was made up with water. The inventor also sets a group of positive control which is composed of

The kit is provided in an RPA nfo reagent kit, and meanwhile, a group of negative controls are also set for the positive controls, the negative controls are not added with a template, and the volume of the template is complemented with water.

Amplification: throwing the magnesium acetate solution on the cover of the reaction tube, fully and uniformly mixing, amplifying for 20min at 37 ℃, taking out the reaction tube for fully and uniformly mixing in the 4 th min, and then putting the reaction tube back to the reaction device for continuous amplification.

And (5) judging a result: mu.L of RPA amplification product was diluted to 100. mu.L with PBST and the RPA product was detected using the lateral flow nucleic acid detection strip described above. One multiple well was set for each reaction.

The test result of the lateral chromatography nucleic acid test strip is shown in figure 1. FIG. 1 is a diagram showing the detection lines and quality control lines of the primer-probe combination, positive control and its corresponding negative control lateral chromatography nucleic acid detection test strip when the detection time is 5 min.

The primer combination determined by the invention comprises: two forward primers PrF1 and two reverse primers PrR2 respectively have oligonucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO. 3.

(3) Determination of the Probe

Probe-1 probes listed in Table 1 are preferred in the present invention, and the length of the probe is 49bp, wherein the 5 'end is 34bp, and the 3' end is 15 bp.

The probe consists of fluorescein (carboxyfluorescein FAM), a 5 ' terminal sequence, Tetrahydrofuran (THF), a 3 ' terminal sequence and a 3 ' terminal extension blocking group (phosphate group). The probe has SEQ ID No.: 4, and the 5 'end is marked with fluorescein FAM, the 3' end is added with an extension blocking group (such as a phosphate group), and Tetrahydrofuran (THF) is inserted between the 34 th base and the 35 th base. The probe is annealed with amplified DNA marked with Biotin, and nfo enzyme in an RPA system cuts the probe at a THF position, so that the probe can continue to extend at the 3' end under the action of polymerase, and finally, an amplification product double-marked by FAM and Biotin is obtained.

Example 2: optimization of RPA reaction systems, amplification and detection conditions

In the process of primer screening, the lateral chromatography nucleic acid detection test strip still has false positive in detection, so that the RPA reaction system, the amplification and detection conditions need to be optimized

(1) Concentration of primer Probe

Setting the concentration gradient of the reverse primer as 10 mu mol/L, 5 mu mol/L and 2.5 mu mol/L, setting the concentration gradient of the probe as 10 mu mol/L, 5 mu mol/L and 2.5 mu mol/L, combining the reverse primer with one concentration with the probes with three concentrations respectively to form 3 groups of combinations, and setting one group of negative control in each group of combinations. Respectively carrying out RPA amplification at 37 ℃, and taking the condition shown by a detection line of a lateral chromatography nucleic acid detection test paper strip as an index after the amplification is finished. The combination with the best amplification effect and no false positive is screened out.

Through analyzing the detection result of the lateral chromatography nucleic acid detection test paper strip, the concentration of the reverse primer determined by the invention is 5 mu mol/L, and the concentration of the probe is 10 mu mol/L.

(2) Time of amplification

A50 μ L RPA reaction was as follows: mu.mol/L forward primer 2.1. mu.L, 5. mu. mol/L reverse primer 2.1. mu.L, 10. mu. mol/L probe 0.6. mu.L, 1X 104 copies/. mu.L template 1. mu.L, 12.2. mu.L DNase-and RNase-free water and 29.5. mu.L buffer were added to 0.2mL of a lyophilized enzyme powder

nfo reaction tube. Then 2.5. mu.L of a 280mM magnesium acetate solution was added to the cap of the reaction tube.

Amplification: throwing the magnesium acetate solution on the cover of the reaction tube, fully and uniformly mixing, amplifying at 37 ℃ for 10min, 15min and 20min, reacting for 4min, taking out the reaction tube, fully and uniformly mixing, and then putting back to the reaction device for continuous amplification. The inventor sets a group of negative controls for each group, the negative controls do not add a template, and the volume of the template is complemented by water.

And (5) judging a result: mu.L of the RPA amplification product was diluted to 100. mu.L with PBST and the RPA product was detected using the lateral flow nucleic acid detection test strip described above.

By analyzing the detection result of the lateral chromatography nucleic acid detection test paper strip, the amplification time of 10min and 15min is shorter, and the amplification time determined by the invention is 20min in consideration of the subsequent sensitivity test.

In conclusion, through optimization of an RPA reaction system and amplification and detection conditions, the finding confirms that the detection effect is best when a downstream primer with the concentration of 5 mu mol/L and a probe with the concentration of 10 mu mol/L are used for amplification for 20min, the sample adding amount is 5 mu L, and the color development time of a test strip is controlled to be 3-5 min.

Example 3: sensitivity evaluation of RPA detection

The positive plasmid was diluted to a series of different concentrations of 104 to 1/. mu.L at a 10-fold ratio, 1. mu.L of each was added to the reaction system determined in example 2, and the RPA detection was carried out on the templates of different copy numbers using the selected primer combinations and the amplification and detection conditions determined in example 2, and the sensitivity of the RPA detection was observed.

As a result: all the samples are positive from 10 copies/. mu.L, which indicates that the sensitivity of the RPA detection method of the invention reaches 10 copies/. mu.L.

Example 4: evaluation of specificity of RPA detection

The genomic DNA of Streptococcus pneumoniae and Streptococcus pyogenes, Streptococcus agalactiae, human plasma DNA were evaluated as controls to determine the specificity of the RPA detection method of the invention.

Respectively taking genome DNA of streptococcus pneumoniae, streptococcus pyogenes and streptococcus agalactiae and human plasma DNA as templates, and adopting the following reaction systems: mu.l of forward primer 2.1. mu.l, 5. mu. mol/L of reverse primer 2.1. mu.l, 10. mu. mol/L of probe 0.6. mu.l, 1. mu.l of sample, 12.2. mu.l of DNase-free and RNase-free water and 29.5. mu.l of buffer were combined to make a premix, which was added to a 0.2mL TwistAmp nfo reaction tube containing lyophilized enzyme powder. Then 2.5. mu.L of a 280mM magnesium acetate solution was added to the cap of the reaction tube.

Amplification: throwing the magnesium acetate solution on the cover of the reaction tube, fully and uniformly mixing, amplifying for 20min at 37 ℃, taking out the reaction tube for reacting for 4min, fully and uniformly mixing, and then putting back to the reaction device for continuous amplification.

And (5) judging a result: diluting 5 μ L of RPA amplification product to 100 μ L with PBST, detecting the RPA product with the lateral chromatography nucleic acid detection test strip, wherein the detection line and the quality control line are both shown, and the judgment result is positive; the detection line is not shown, the quality control line is shown, and the judgment result is a negative result; the quality control line is not shown, and whether the detection line is shown or not is judged that the result is invalid.

As a result: referring to fig. 2, the detection lines of the genomic DNA of streptococcus pyogenes and streptococcus agalactiae and the human plasma DNA sample are both negative, and only the detection line of the streptococcus pneumoniae sample is clear and positive, which shows that the RPA detection method of the present invention has strong specificity to streptococcus pneumoniae.

The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.

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Claims

1. A non-diagnostic method for detecting streptococcus pneumoniae based on RPA, comprising the steps of:

2. The RPA-based non-diagnostic method for the detection of streptococcus pneumoniae as claimed in claim 1, wherein fluorescein is carboxyfluorescein FAM or FITC fluorescein, and the extension blocking group is a phosphate group.

3. The RPA-based non-diagnostic method for the detection of streptococcus pneumoniae according to claim 1, wherein the probe has a length of 49bp, wherein the 5 'end is 34bp, and the 3' end is 15 bp.

4. The RPA-based non-diagnostic method for detecting Streptococcus pneumoniae according to claim 1, wherein in S1, a primer set and a probe are used in a 50 μ L RPA reaction system to perform RPA reaction, the concentration of the forward primer is 10 μmol/L, the concentration of the reverse primer is 5 μmol/L, the concentration of the probe is 10 μmol/L, the concentration of magnesium ions is 280 μmol/L, and the sample adding amount of the template is 1 μ L; a premix of 2.1. mu.L of the forward primer, 2.1. mu.L of the reverse primer, 0.6. mu.L of the probe, 1. mu.L of the sample, 12.2. mu.L of DNase-free and RNase-free water and 29.5. mu.L of buffer was added to a 0.2mL TwistAmp nfo reaction tube containing lyophilized enzyme powder, and then 2.5. mu.L of a magnesium acetate solution was added to the cap of the reaction tube; throwing the magnesium acetate solution on the cover of the reaction tube, fully and uniformly mixing, amplifying at 37 ℃ for 20min, taking out the reaction tube for fully and uniformly mixing in the 4 th min of reaction, and then putting the reaction tube back to the reaction device for continuous amplification; in S2, the sample adding amount is 5 μ L during detection, and the color development time of the test strip is controlled within 3-5 min.

5. A kit for detecting and detecting streptococcus pneumoniae is characterized by comprising a primer group and a probe; the primer group and the probe are designed according to a streptococcus pneumoniae specific conserved sequence, the streptococcus pneumoniae specific conserved sequence has a nucleotide sequence shown in SEQ ID No.1, a forward primer in the primer group has an oligonucleotide sequence shown in SEQ ID No.2, a reverse primer has an oligonucleotide sequence shown in SEQ ID No.3, and the 5' end is marked with biotin; the probe has an oligonucleotide sequence shown in SEQ ID NO.4, fluorescein is marked at the 5 'end, an extension blocking group is added at the 3' end, and tetrahydrofuran is added between 34 th and 35 th bases.

6. An RPA special primer for detecting streptococcus pneumoniae is designed according to a streptococcus pneumoniae specific conserved sequence, and the streptococcus pneumoniae specific conserved sequence has a nucleotide sequence shown in SEQ ID NO. 1; the forward primer in the primers has an oligonucleotide sequence shown in SEQ ID NO.2, the reverse primer has an oligonucleotide sequence shown in SEQ ID NO.3, and the 5' end is marked with biotin.

7. An RPA probe for detecting streptococcus pneumoniae, which is designed according to a streptococcus pneumoniae-specific conserved sequence having a nucleotide sequence shown in SEQ ID No. 1; the probe has an oligonucleotide sequence shown in SEQ ID NO.4, fluorescein is marked at the 5 'end, an extension blocking group is added at the 3' end, and tetrahydrofuran is added between 34 th and 35 th bases.

8. Non-diagnostic use of the kit according to claim 5 for the detection of streptococcus pneumoniae.

9. Non-diagnostic use of the RPA specific primers according to claim 6 for the detection of streptococcus pneumoniae.

10. Non-diagnostic use of the RPA probe of claim 7 for the detection of streptococcus pneumoniae.