CN114540525A - Primer composition for detecting or assisting in detecting chlamydia psittaci and application thereof - Google Patents

Abstract

The invention discloses a composition for detecting or assisting the detection of Chlamydia psittaci and application thereof, and belongs to the field of compositions used in detection methods comprising nucleic acids or microorganisms. The technical problem to be solved by the present invention is how to detect or assist the detection of Chlamydia psittaci with high sensitivity and/or high specificity and/or fast/or simple. The present invention provides a primer composition for detecting or assisting the detection of Chlamydia psittaci, the primer composition is composed of single-stranded DNA molecules whose nucleotide sequences are respectively shown in Sequences 2 and 3 of the Sequence Listing. Using the composition of the present invention for detection or auxiliary detection of Chlamydia psittacosis with RAA amplification to detect Chlamydia psittacosis is fast and efficient, and the amplification reaction can be completed within 15 minutes, and only a large amount of amplification reaction can be completed at a constant temperature The detection results have good specificity and high sensitivity; the identification method is simple and convenient, and is suitable for rapid detection of field or clinical specimens.

Description

A kind of primer composition for detecting or assisting in detecting Chlamydia psittaci and its application

technical field

The present invention relates to a primer composition for detecting or assisting the detection of Chlamydia psittaci and its application in the field of compositions used in the detection method comprising nucleic acid or microorganism.

Background technique

Chlamydia psittaci ( Chlamydia psittaci ) belongs to the order Chlamydiales ( Chlamydiales ), Chlamydiaceae ( Chlamydiaceae ), Chlamydia ( Chlamydia ), a Gram-negative, strictly intracellular parasitic microorganism, the size is between bacteria and viruses, It is mainly transmitted by air and contact, and is a kind of biological warfare agent. Parrots were initially thought to be the host of the pathogen, and the disease caused by it was called psittacosis, and humans, birds and some mammals are susceptible. Birds or poultry with bacteria, bacteria-containing secretions or excreta contaminated environment, feathers and dust can be the source of infection. Human infection with psittacosis is usually characterized by high fever, aversion to cold, headache, myalgia, cough, etc. 85% to 90% of patients develop pneumonia, and severe patients can be complicated by myocarditis, endocarditis, and pulmonary edema.

Serological diagnosis is mainly based on indirect immunofluorescence. However, this method can only detect Chlamydia psittacosis antibodies 1-2 weeks after the onset of the disease, and the timeliness is poor. Gene detection technology based on the principle of PCR is the mainstream method of molecular biology diagnosis of Chlamydia psittacosis, but the detection requires the use of professional instruments, the reaction time is also long, and the detection sensitivity needs to be improved, and it cannot adapt to the clinical "simple, fast" , sensitive" requirements.

Recombinase-aided Amplification (RAA) can complete a large number of trace nucleic acid amplifications in a short time under isothermal conditions. It is a "simple, fast, sensitive and accurate" gene suitable for the field Amplification detection method. In RAA technology, primers and probes are the key factors that determine the sensitivity and specificity of detection results.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is how to detect Chlamydia psittaci with high sensitivity and/or high specificity and/or quickly/or simply.

In order to solve the above technical problems, the present invention provides the following technical solutions.

A first object of the present invention is to provide a primer composition for detecting Chlamydia psittaci, consisting of forward primer F1 and reverse primer R3;

The forward primer F1 is as follows (a1) or (a2):

(a1) the single-stranded DNA molecule shown in Sequence 2 of the Sequence Listing;

(a2) a DNA molecule having the same function as sequence 2 by substitution and/or deletion and/or addition of one or several nucleotides in sequence 2;

The reverse primer R3 is as follows (a3) or (a4):

(a3) the single-stranded DNA molecule shown in Sequence 3 of the Sequence Listing;

(a4) A DNA molecule having the same function as sequence 3 by substitution and/or deletion and/or addition of one or several nucleotides in sequence 3.

The 5' end of the reverse primer was labeled with Biotin. The double-stranded DNA obtained after the amplification of the forward and reverse primers is completed will be labeled with biotin.

Described primer is designed according to the conserved gene CPSIT_RS02830 of Chlamydia psittacosis, through software, the gene homologous sequence is comparatively analyzed and combined with the RAA amplification fragment optimum length, further confirmed the RAA amplification target sequence of this gene of Chlamydia psittacosis: The 29th to 258th nucleotide sequence, this region contains a nucleotide fragment of 230 bases, and has the nucleotide sequence shown in SEQ ID NO: 1.

The present invention also provides a primer-probe composition, which consists of the above-mentioned primer composition and probe;

The probe has the oligonucleotide sequence shown in sequence 4, and is labeled with fluorescein at the 5' end, an extension blocking group is added at the 3' end, and a tetrahydrofuran is added between the 30th and 31st bases.

The fluorophore is FAM or FITC.

The extension blocking group is a phosphate group.

The probe has the oligonucleotide sequence shown in sequence 4, and the 5' end is labeled with fluorescein FAM, and the 3' end is added with an extension blocking group (such as a phosphate group), between the 30th and 31st bases. Add a tetrahydrofuran (THF) in between. The probe is annealed to the amplified DNA labeled with biotin, and the nfo enzyme in the RAA system will cut the probe at the THF site, so that the probe can continue to extend at the 3' end under the action of polymerase, and finally obtain FAM and Biotin double-labeled amplification products.

The present invention also provides a detection reagent for detecting or assisting the detection of Chlamydia psittaci, the detection reagent includes the above-mentioned primer-probe composition.

The molar ratio of the forward primer F1, the reverse primer R3 and the probe in the detection reagent is 1:1:0.3.

The concentrations of primers and probes in the detection reagents were both 2.5 μM.

The present invention also provides a kit for detecting or assisting in the detection of Chlamydia psittaci, the kit contains the primer composition or the primer-probe composition or the detection reagent.

The application of the above-mentioned primer composition in the preparation of a product to determine whether the sample to be tested is infected or contains Chlamydia psittaci should also fall within the protection scope of the present invention.

The application of the above-mentioned primer-probe composition in the preparation of a Chlamydia psittacosis detection reagent or kit should also fall within the protection scope of the present invention.

The application of the detection reagent in the preparation of the Chlamydia psittacosis detection reagent or kit should also fall within the protection scope of the present invention.

The present invention also relates to a RAA detection method for the rapid detection of Chlamydia psittacosis, the reagent for the RAA reaction system of 50 μL in the RAA detection method for the rapid detection of Chlamydia psittacosis, the concentration of the forward primer is 2.5 μM, the concentration of reverse primer is 2.5 μM, and the concentration of probe is 2.5 μM; combine 2 μL forward primer, 2 μL reverse primer, 0.6 μL probe, 5 μL sample, 1.4 μL DNase and RNase-free water and 38 μL solution A premix solution was added to a 0.2 mL RAA-nfo reaction tube containing lyophilized enzyme powder, and then 1 μL of solution B was added to the lid of the reaction tube; the solution B on the lid of the reaction tube was shaken off and mixed well After amplification at 39°C for 15 min, the sample volume was 5 μL during detection, and the color development time of the test strip was controlled within 3 min.

The present invention provides an RAA detection method for the rapid detection of Chlamydia psittacosis, which adopts the above-mentioned RAA primers and probes to amplify, and combines with lateral chromatography nucleic acid detection test strips for visual judgment.

The RAA detection method for the rapid detection of Chlamydia psittaci of the present invention comprises the following steps:

(1) Using the genomic DNA of the sample to be tested as a template, carry out the RAA reaction under the labeling of the primer set and the probe;

(2) Judgment of results: The RAA product was detected with lateral chromatography nucleic acid detection test strips. Both the detection line and the quality control line were displayed, and the judgment result was a positive result; the detection line was not displayed, but the quality control line was displayed. The judgment result is a negative result; the quality control line is not displayed, regardless of whether the test line is displayed, the result is judged to be invalid.

The RAA detection method for the rapid detection of Chlamydia psittaci according to the present invention, the concrete steps are as follows:

(1) Amplification reagent preparation and sample loading: 2.5 μM forward primer 2 μL, 2.5 μM reverse primer 2 μL, 2.5 μM probe 0.6 μL, 5 μL sample, 1.4 μL DNase and RNase-free water, and 38 μL solution A to form a master mix , added to a 0.2 mL RAA-nfo reaction tube containing lyophilized enzyme powder. Then add 1 μL of solution B to the lid of the reaction tube;

(2) Amplification: shake off the solution B on the lid of the reaction tube, mix well and amplify at 39°C for 15 minutes;

(3) Judgment of results: Dilute 5 μL of RAA amplification product with PBS to 100 μL, and use lateral flow test strips to detect the recovered RAA product. Both the detection line and the quality control line are displayed, and the 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, regardless of whether the detection line is shown, the result is judged to be invalid.

The RAA detection method for the rapid identification of Chlamydia psittaci of the present invention detects the specific conserved target sequence of Chlamydia psittaci, that is, the conserved sequence of Chlamydia psittaci CPSIT_RS02830, which can be used as one of the marker genes of Chlamydia psittacosis.

The RAA detection method for the rapid detection of Chlamydia psittaci of the present invention saves the detection time of Chlamydia psittacosis, the amplification reaction can be completed within 15 minutes at 39 DEG C, and the entire detection process can be completed within 20 minutes, which is comparable to conventional PCR and Real-time PCR significantly reduces detection time compared to hours.

The RAA detection method for the rapid detection of Chlamydia psittaci of the present invention reduces the reaction temperature, and the RAA only needs a constant temperature of 39°C to complete the experiment, which is far lower than the 60-95°C of ordinary PCR and fluorescence quantitative PCR and the ring-mediated temperature. 63°C for loop-mediated isothermal amplification (LAMP).

The RAA detection method for rapid detection of Chlamydia psittaci of the present invention is simpler and easier to carry: the enzymes required for amplification and some other necessary things can be lyophilized and stored, and can be stored at room temperature for a long time, and the sample does not need to be processed complex reaction.

The RAA detection method for the rapid detection of Chlamydia psittaci of the present invention has high sensitivity and strong specificity. It can be used for field or clinical detection and has broad application prospects.

Experiments of the present invention prove that the primers and probe compositions designed and synthesized by the inventors adopt the RAA method to specifically detect Chlamydia psittaci. The advantages are as follows: (1) A large number of amplification reactions can be completed only at a constant temperature, without the need for Professional PCR equipment; (2) High specificity, can accurately detect Chlamydia psittacosis, and the experimental results show that the Parasite, Rickettsia australis, Rickettsia Przewalskii, Rickettsia Przewalskii, Henzerling strain of C. bainii, seven strains of C. bainii, Legionella pneumophila, Chaffee Rickettsia, Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Vibrio cholerae, Bartonella 5 days, Salmonella typhi, Streptococcus suis, Shigella sonnei, Chlamydia trachomatis did not cross-react (3) High sensitivity, the minimum detection limit is ¹ ×100 copies/μL; (4) Fast and efficient, the amplification reaction can be completed within 15 minutes; (5) The identification method is simple and convenient, within 3 minutes after amplification The results can be observed with the naked eye, which is suitable for on-site detection of clinical specimens.

Description of drawings

Figure 1 shows the detection results of different primer and probe combinations in the optimal primer and probe combination screening of the RAA detection system.

Fig. 2 is to determine the optimal amplification temperature and time of the detection method for the detection of Chlamydia psittaci RAA.

Figure 3 shows the sensitivity of the method for detecting Chlamydia psittaci RAA.

Figure 4 shows the specificity of the method for detecting Chlamydia psittaci RAA.

Figure 5 shows the reproducibility of the RAA method for detection of Chlamydia psittaci genomic DNA.

Figure 6 shows the detection of Chlamydia psittaci infected mouse samples by RAA method.

Figure 7 shows the sensitivity of fluorescence quantitative PCR to detect Chlamydia psittaci.

Figure 8 shows primer and probe sequences.

Figure 9 shows the combination of 18 primer-probe sets.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product specification. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

DNeasy Blood & Tissue Kit Whole Genome Extraction Kit in the following examples: Qiagen Company, Cat. No. 69506.

RAA-nfo nucleic acid amplification kit (test strip type): Hangzhou Zhongce Biotechnology Co., Ltd., product number S005ZC.

Milenia GenLine HybriDetect lateral flow test strip: Milenia Biotec, Germany, product number MGHD 1.

Thermostatic metal bath: Cayudi, model: H2O3-PRO.

Some of the materials in the following examples are as follows.

Chlamydia psittaci (strain 6BC, named Chlamydophila psittaci in the literature) is disclosed in the document "Genotyping of Chlamydophila psittaci strains derived from avian andmammalian species. Vet Res Commun. 2009 Aug;33(6):577-80."

The genomic DNA of Canadian rickettsia was published in the document "Research on the detection of rickettsia by broad-spectrum primer PCR method, Chinese Journal of Zoonosis, 1994, 10(02): 25-27", Chlamydia trachomatis genomic DNA Published in the document "Cloning and expression of polymorphic membrane protein D gene of Chlamydia trachomatis and its neutralization in immune serum, Advances in Microbiology and Immunology, Vol. 37, No. 1, 11-15, 2009", Ricketts Australia (Phillips) (hereinafter referred to as Rickettsia australis) genomic DNA was established in the literature "Real-time fluorescence quantitative PCR detection method of Rickettsia speckle fever, PLA Medical Journal, Vol. 33, No. 11, 1297-1299, 2008 It was disclosed in the "Anaplasma phagocytosis genomic DNA, Salmonella typhimurium genomic DNA, Listeria monocytogenes genomic DNA, Bartonella 5 day fever genomic DNA, Salmonella typhi genomic DNA, Coxodia strain, Henzerling strain) genomic DNA, Shigella songnei genomic DNA, Streptococcus suis genomic DNA, Staphylococcus aureus genomic DNA, Vibrio cholerae genomic DNA, Rickettsia rickettsia genomic DNA, Heilongjiang Rickettsia genome DNA, Rickettsia Siberia genomic DNA, Rickettsia Przewalskii genomic DNA, Rickettsia morseii genomic DNA, Legionella pneumophila genomic DNA, Ehrlichia chaffei genomic DNA in "real-time quantitative PCR Rapid Detection of 4 Types of Pathogenic Rickettsia, Acta Parasites and Medical Entomology, Vol. 26, No. 2, 110-117, 2019.”; publicly available from the Academy of Military Medicine, Academy of Military Sciences of the Chinese People’s Liberation Army .

The following examples are only intended to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are usually based on conventional conditions such as those described in the "Molecular Cloning Experiment Guide" edited by Sambrook et al., or according to the conditions suggested by the manufacturer.

The ways of obtaining various biological materials described in the examples are only to provide an experimental way to achieve the purpose of specific disclosure, and should not be a limitation on the source of the biological materials of the present invention. In fact, the sources of biological materials used are extensive, and any biological materials that can be obtained without violating laws and ethics can be replaced and used according to the tips in the examples.

The RAA primers and probes used were synthesized by Nanjing GenScript Biotechnology Co., Ltd., and the qPCR primers and probes were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

Example 1. Design and screening of Chlamydia psittaci primers and probes.

(1) Design of primers and probes.

Through file retrieval, the inventors analyzed and determined that the specific sequence in the CPSIT_RS02830 gene of Chlamydia psittaci in the present invention is the target gene. The known template gene sequence, that is, the nucleotide sequence shown in SEQ ID NO: 1, was obtained from the NCBI database. According to the RAA primer and probe design principles, 6 primers and 2 probes were designed, and the sequences are shown in Figure 8. The probe consists of a fluorescein (such as carboxyfluorescein FAM), a 5'-end sequence, tetrahydrofuran (THF), a 3'-end sequence, and a 3'-end extension blocking group (such as a phosphate group). The length of the probe is 45-60 bp, of which the 5' end is at least 30 bp and the 3' end is at least 15 bp.

The probe of the present invention is labeled with fluorescein FAM at the 5' end, and an extension blocking group (such as a phosphate group) is added at the 3' end, and a tetrahydrofuran (THF) is added between the 30th and 31st bases. The probe is annealed to the amplified DNA labeled with biotin, and the nfo enzyme in the RAA system will cut the probe at the THF site, so that the probe can continue to extend at the 3' end under the action of polymerase, and finally obtain FAM and Biotin double-labeled amplification products.

(2) Primer and probe screening.

The DNeasy Blood & Tissue Kit Whole Genome Extraction Kit (Qiagen Company, Cat. No. 69506) was used to extract the genomic DNA of Chlamydia psittaci in cell culture, and the extracted genomic DNA was quantitatively detected by fluorescence. The specific method is as follows: using the genomic DNA as a template, the primers and probes are combined into 18 sets of primers and probes, and the combination numbers are shown in FIG. 9 . 18 sets of primers and probe sets were respectively carried out for RAA amplification at 39°C for 15 minutes according to the instructions, and the primers with the highest amplification efficiency under the condition of 39°C were screened out with the detection line of the lateral flow test strip as an indicator. Probe combinations for the evaluation and application of subsequent RAA detection.

The primer probe of fluorescent quantitative PCR is: primer Cp-F'-GGTTCCGCTCTCTCCTTACAAG-3' (sequence 10); primer Cp-R'-CCCACATAGTGCCATCGAT-3' (sequence 11); probe TaqMan MGB-probe 5'-TGCCTGTAGGGAACCCAGCTGAAC- 3' (sequence 12).

The 50 μL RAA reaction system for screening is as follows: 2.5 μM forward primer 2 μL, 2.5 μM reverse primer 2 μL, 2.5 μM probe 0.6 μL, 1×10 ⁵ copies/μL template 5 μL, 1.4 μL DNase and RNase-free water and 38 μL of solution A (Hangzhou Zhongtest Biotechnology Co., Ltd., Product No. S005ZC) to form a premix solution, which was added to the reaction tube of 0.2 mL of RAA-nfo kit containing lyophilized enzyme powder. Then add 1 μL of solution B (Hangzhou Zhongtest Biotechnology Co., Ltd., product number S005ZC) to the lid of the reaction tube, and set up a set of negative controls. The negative control does not add template, and the template volume is supplemented with water. Amplification: shake off the solution B on the lid of the reaction tube, mix well and amplify at 39°C for 20min. Judgment of results: Take 5 μL of RAA amplification product and dilute it with PBS to 100 μL, and then use lateral flow test strip to detect the RAA product. One duplicate well was set up for each reaction.

Figure 1 shows the detection results of colloidal gold lateral chromatography nucleic acid detection test strips for 18 sets of primer combinations. Figure 1 shows the display of the lateral flow test strip detection lines and quality control lines of 18 groups of primers, probe combinations and negative controls when the reaction time is 30 min and the detection time is 3 min.

The numbers 1-18 in A in Fig. 1 are the amplification results using the primer-probe combinations numbered 1-18 in Table 2, respectively, and 19 is the NC negative control. The brightest 1, 12, 14, and 17 were further screened according to the above conditions. The results are shown in B in Figure 1. The numbers 1-4 in B correspond to primer probe combinations 1, 12, 14, and 17 for 5 minutes of amplification. 5-8 correspond to primer-probe combinations 1, 12, 14, and 17 for 10 minutes of amplification, 9-12 correspond to primer-probe combinations 1, 12, 14, and 17 for 15 minutes of amplification, and 13 is NC Negative control; it is found that the primer-probe combination numbered 12 has the best detection effect, and the primers in the primer-probe combination numbered 12 include forward primer F1 and reverse primer R3, a total of two, respectively with such as The oligonucleotide sequences shown in Sequence 2 and Sequence 3 are the 29-60 nucleotide sequence and the 226-258 nucleotide sequence of the conserved gene CPSIT_RS02830 of Chlamydia psittaci respectively, wherein the reverse primer is 5' End-labeled with Biotin. Described probe is the nucleotide sequence of the 111th to 157th position of the conserved gene CPSIT_RS02830 of Chlamydia psittacosis, has the oligonucleotide sequence shown in sequence 4, and the 5' end is labeled with fluorescein, and the 3' end is added to block the extension group, adding a tetrahydrofuran (THF) between bases 30-31.

(3) Amplification temperature screening.

The 50 μL RAA reaction system is as follows: 2.5 μM forward primer F1 2 μL, 2.5 μM reverse primer R3 2 μL, 2.5 μM probe probe2 0.6 μL, 1×10 ⁵ copies/μL template 5 μL, 1.4 μL DNase and RNase-free water and 38 μL of buffer A (Hangzhou Zhongtest Biotechnology Co., Ltd., product number S005ZC) to form a premix solution, which was added to a 0.2 mL RAA-nfo reaction tube containing lyophilized enzyme powder. Then 1 μL of solution B (Hangzhou Zhongce Biotechnology Co., Ltd., Cat. No. S005ZC) was added to the lid of the reaction tube. Amplification: shake off the solution B on the lid of the reaction tube, mix well and set the amplification temperature at 37°C, 38°C, 39°C, 40°C, 41°C, and 42°C for 15 minutes respectively. A set of negative controls were set up. The negative controls did not add template, and the template volume was supplemented with water. Judgment of results: Take 5 μL of RAA amplification product and dilute it with PBS to 100 μL, and then use lateral flow test strip to detect the RAA product.

The results are shown in A in Figure 2. Numbers 1-6 in A in Figure 2 correspond to the amplification results at the amplification temperatures of 37°C, 38°C, 39°C, 40°C, 41°C, and 42°C, respectively, and 7 represents NC Negative control; through the analysis of the detection results of A in Figure 2, it is found that there is no sample detection line band at the amplification time of 37 ° C, the sample detection line band is visible to the naked eye at 38 ° C, and the line is the deepest at 39 ° C, so the present invention determines The optimal amplification temperature is 39 °C.

(4) Screening of amplification time.

The 50 μL RAA reaction system is as follows: 2.5 μM forward primer F1 2 μL, 2.5 μM reverse primer R3 2 μL, 2.5 μM probe probe2 0.6 μL, 1×10 ⁵ copies/μL template 5 μL, 1.4 μL DNase and RNase-free water Prepare a premix with 38 μL of buffer A and add it to a 0.2 mL RAA-nfo reaction tube containing lyophilized enzyme powder. Then 1 μL of solution B was added to the lid of the reaction tube. Amplification: Shake off the solution B on the lid of the reaction tube, mix thoroughly, and amplify at 39°C for 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min. A set of negative controls were set up. The negative controls did not add template, and the template volume was supplemented with water. Judgment of results: Take 5 μL of RAA amplification product and dilute it with PBS to 100 μL, and then use lateral flow test strip to detect the RAA product.

The results are shown in B in Figure 2. Numbers 1-6 in B in Figure 2 correspond to amplification results with amplification times of 5min, 10min, 15min, 20min, 25min, and 30min, respectively, and 7 represents the NC negative control; through Figure 2 In the analysis of the detection results of B, the primer-probe combination has no sample detection line band at the amplification time of 5min, the sample detection line band is visible to the naked eye at the amplification time of 10min, and the sample detection line band is colored at the amplification time of 30min. The deepest, considering the follow-up sensitivity test and relatively fast detection time, in view of the fact that 10min can just detect the band, and the detection time of 30min is too long, so the selected 15min is selected, which can not only meet the detection, but also meet the fast detection time. requirements, that is, the optimal amplification time determined by the present invention is 15min.

Example 2: Sensitivity evaluation of RAA detection.

The genomic DNA of Chlamydia psittaci was diluted ¹⁰ times to a series of different concentrations ^ranging from 105 to 100 copies/μL, and 5μL was added to the reaction system determined in Example 1 (that is, the reaction temperature was 39°C and the amplification time was 15min). ), using the selected primer probe set (the primer probe set No. 12), using the amplification and detection conditions determined in Example 1 to perform RAA detection on the above templates with different copy numbers, and observe the sensitivity of RAA detection. A set of negative controls were set up. The negative controls did not add template, and the template volume was supplemented with water.

As a result, the results are shown in Figure 3. The template concentrations of lanes numbered 1-6 in Figure 3 are 1×10 ⁵ copies/μL, 1×10 ⁴ copies/μL, 1×10 ³ copies/μL, 1 ×10 ² copies/μL, 1×10 ¹ copies/μL, 1×10 ⁰ copies/μL, 7 represents NC negative control; as can be seen from Figure 3, starting from 1×10 ⁰ copies/μL The above samples were all positive, indicating that the sensitivity of the RAA detection method of the present invention reached ¹ ×100 copies/μL.

Example 3: Specificity evaluation of RAA detection.

Specificity was evaluated by Anaplasma phagocytogenes, Rickettsia rickettsiae, Rickettsia Heilongjiang, Rickettsia Siberia, Rickettsia Canada, Rickettsia australis, Rickettsia Morsei, Rickettsia Platt. Rickettsia, Henzerling strain of C. bainii, seven strains of C. bainii, Legionella pneumophila, Ehrlichia chaffee, Listeria monocytogenes, Salmonella typhimurium, aureus The genomic DNAs of Staphylococcus, Vibrio cholerae, Bartonella 5 days, Salmonella typhi, Streptococcus suis, Shigella sonnei and Chlamydia trachomatis were used as controls to determine the specificity of the RAA detection method of the present invention.

Anaplasma phagocytosis, Rickettsia rickettsii, Rickettsia Heilongjiang, Rickettsia Siberia, Rickettsia Canada, Rickettsia australis, Rickettsia Przewalskii, Rickettsia Przewalskii Chlorella, Henzerling strain of C. bainii, seven strains of C. bainii, Legionella pneumophila, Ehrlichiella chaffee, Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus , Vibrio cholerae, Bartonella 5 days, Salmonella typhi, Streptococcus suis, Shigella sonei and Chlamydia trachomatis genomic DNA as templates, and 5 μL of each was added to the reaction system determined in Example 1, using primer combination 12 , using the amplification and detection conditions determined in Example 1 to perform RAA detection on the above-mentioned different templates to observe the specificity of RAA detection. A set of negative controls were set up. The negative controls did not add template, and the template volume was supplemented with water, which was represented by NC; 5 μL of Chlamydia psittacosis was taken for the same detection, which was represented by PC.

The results are shown in Figure 4. The templates numbered 1-21 in Figure 4 are Anaplasma phagocytophila, Rickettsia Heilongjiang, Rickettsia rickettsii, Rickettsia Siberia, Rickettsia Canada, and Rickettsia australis. rickettsia Genomic DNA of Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Vibrio cholerae, Bartonella 5 days, Salmonella typhi, Streptococcus suis, Shigella sonnei, Chlamydia trachomatis, 22 were NC negative Control, 23 is the genomic DNA of Chlamydia psittaci. Figure 4 shows: Anaplasma phagocytosis, Rickettsia rickettsii, Rickettsia Heilongjiang, Rickettsia Siberia, Rickettsia Canada, Rickettsia Australia, Rickettsia Morse, Rickettsia Przewalski, Henzerling strain of C. bainii, seven strains of C. bainii, Legionella pneumophila, Ehrlichia chaffee, Listeria monocytogenes, Salmonella typhimurium, No bands were found on the genomic DNA of Staphylococcus aureus, Vibrio cholerae, Bartonella 5 days, Salmonella typhi, Streptococcus suis, Shigella sonnei, and Chlamydia trachomatis (numbered 1-21 in the figure). , was negative, only the Chlamydia psittacosis sample (numbered as 23 in the figure) showed a clear band in the detection line, which was positive, indicating that the RAA detection method of the present invention has strong specificity to Chlamydia psittacosis.

Example 4. Repeatability evaluation of RAA detection.

Take 5 μL of Chlamydia psittaci DNA (1×10 ⁵ copies/μL) and add it to the reaction system determined in Example 1 (that is, the reaction temperature is 39 °C and the amplification time is 15 min), using primer combination 12, using the amplification determined in Example 1. The RAA detection was performed on the template according to the detection conditions, and the detection was repeated 6 times to observe the repeatability of the RAA detection. A set of negative controls were set up. The negative controls did not add template, and the template volume was supplemented with water.

The results are shown in Figure 5. Nos. 1-6 in Figure 5 were tested with 1×10 ⁵ copies/μL of psittacosis genomic DNA as the template, and 7 was the negative control for NC; all 6 repeated detection lines appeared The clear band is positive, indicating that the RAA detection method of the present invention has good repeatability.

Example 5. RAA detection of Chlamydia psittaci infection of experimental animal samples.

Infect mice with Chlamydia psittaci in the above example, take 5 μL of Chlamydia psittaci-infected mouse spleen tissue DNA (respectively 1×10 ⁵ copies/μL) and add it to the reaction system determined in Example 1, using primer combination 12 , using the amplification and detection conditions determined in Example 1 (that is, the amplification conditions with the reaction temperature of 39 °C and the amplification time of 15 min) to perform RAA detection on the template, and observe the results of RAA detection of Chlamydia psittaci infection of experimental animal samples. A set of negative controls were set up. The negative controls did not add template, and the template volume was supplemented with water.

The results are shown in FIG. 6 . Nos. 1-3 in FIG. 6 are the experiments using 1×10 ⁵ copies/μL of psittacosis genomic DNA as the template, and 4 is the negative control for NC. Only the detection line of the spleen sample of Chlamydia psittacosis infected mice showed clear bands, indicating that the RAA detection method of the present invention can be used for the detection of Chlamydia psittacosis infected samples.

Comparative Example 1: Fluorescence quantitative PCR amplification of Chlamydia psittaci.

The present invention takes the Chlamydia psittacosis genomic DNA in Example 1 as a template, and uses fluorescence quantitative PCR to detect the Chlamydia psittacosis DNA. Fluorescence quantitative PCR primer sequences are upstream Cp-F'-GGTTCCGCTCTCTCCTTACAAG-3', downstream Cp-R'-CCCACATAGTGCCATCGAT-3', probe TaqMan MGB-probe 5'-TGCCTGTAGGGAACCCAGCTGAAC-3'. A negative control was set, and the template was not added to the negative control, and the volume of the template was supplemented with water.

The results are shown in Figure 7, where A in Figure 7 is the analysis result of the standard curve R ² , wherein the slope (slope) is -6.725051, the intercept (intercept) is 67.729729, and the R ² is 0.999427. In Figure 7, the template in B is Chlamydia psittaci DNA, A6 and A7 represent the template concentration of 1×10 ⁵ copies/μL, B6 and B7 represent the template concentration of 1×10 ⁴ copies/μL, and C6 and C7 represent the template concentration is 1×10 ³ copies/μL, D6, D7 indicate that the template concentration is 1×10 ² copies/μL, E6, E7 indicate that the template concentration is 1×10 ¹ copies/μL, F6, F7 indicate that NTC is a negative control , Undet means no detection result; it can be seen that there are PCR products amplified in lanes A6, A7 and B6, B7, indicating that the sensitivity of ordinary fluorescence quantitative PCR to detect Chlamydia psittaci is 1×10 ⁴ copies/μL.

Therefore, for the same batch of samples, the F1, R3 and probe2 primers and probe combinations of Example 1 are more sensitive to RAA amplification than fluorescence quantitative PCR amplification.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention has given particular embodiments, it should be understood that the present invention can be further modified. In conclusion, in accordance with the principles of the present invention, the present invention is intended to include any alterations, uses or improvements of the present invention, including changes made by conventional techniques known in the art, departing from the disclosed scope of the present invention.

sequence listing

<110> Academy of Military Medicine, Academy of Military Sciences, Chinese People's Liberation Army

<120> A kind of primer composition for detection or auxiliary detection of Chlamydia psittaci and its application

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 230

<212> DNA

<213> Artificial Sequence

<400> 1

gtatagcagc attagccagc gctttagacg aatacgagtc ctccctaaat cagaatgaca 60

caccacaaag acctggcgag tacacggttt catcatcaaa tgacgcagtg tttttgctca 120

gagatataga aaagttaaca gaagttatc atacacttca cttcacactt cctagctggc 180

tgatcaatga tatcaaagat atcgaaaatg gcgcaaaacg tgtgctagaa 230

<210> 2

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 2

gtatagcagc attagccagc gctttagacg a 31

<210> 3

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 3

ttctagcaca cgttttgcgc cattttcgat atc 33

<210> 4

<211> 47

<212> DNA

<213> Artificial Sequence

<400> 4

cacggtttca tcatcaaatg acgcagtgtt tttgctcaga gatatag 47

<210> 5

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 5

tagacgaata cgagtcctcc ctaaatcaga atg 33

<210> 6

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 6

gctttagacg aatacgagtc ctccctaaat ca 32

<210> 7

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 7

cacacgtttt gcgccatttt cgatatcttt ga 32

<210> 8

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 8

gcatttgtga aacttctagc acacgttttg cg 32

<210> 9

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 9

ctaaatcaga atgacacacc acaaagacct ggcgagtaca cggtttca 48

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 10

ggttccgctc tctccttaca ag 22

<210> 11

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 11

cccacatagt gccatcgat 19

<210> 12

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 12

tgcctgtagg gaacccagct gaac 24

Claims (10)

1. a primer composition for detecting or assisting in the detection of Chlamydia psittaci, consists of forward primer F1 and reverse primer R3; The forward primer F1 is as follows (a1) or (a2): (a1) the single-stranded DNA molecule shown in Sequence 2 of the Sequence Listing; (a2) a DNA molecule having the same function as sequence 2 by substitution and/or deletion and/or addition of one or several nucleotides in sequence 2; The reverse primer R3 is as follows (a3) or (a4): (a3) the single-stranded DNA molecule shown in Sequence 3 of the Sequence Listing; (a4) A DNA molecule having the same function as sequence 3 with the substitution and/or deletion and/or addition of one or several nucleotides in sequence 3. 2. a primer-probe composition for detection or auxiliary detection of Chlamydia psittaci, is made up of the described primer composition of claim 1 and probe; Said probe has the oligonucleotide sequence shown in sequence 4 , and the 5' end is labeled with fluorescein, the 3' end is added with an extension blocking group, and a tetrahydrofuran is added between the 30th and 31st bases. 3. The primer-probe composition of claim 2, wherein the fluorescent group is FAM or FITC. 4. The primer-probe composition according to claim 2 or 3, wherein the extension blocking group is a phosphate group. 5. The detection reagent for detecting or assisting in the detection of Chlamydia psittaci, characterized in that the detection reagent comprises the primer-probe composition described in any one of claims 2-4. 6. The detection reagent according to claim 5, wherein the molar ratio of the forward primer F1, the reverse primer R3 and the probe is 1:1:0.3. 7. a test kit for detection or auxiliary detection of Chlamydia psittaci, is characterized in that, described test kit contains primer composition described in claim 1 or contains the primer probe combination described in any one of claim 2-4 or contain the detection reagent according to claim 5 or 6. 8. the application of the primer composition described in claim 1 in the preparation of Chlamydia psittaci detection reagent or test kit. 9. the application of the primer-probe composition described in any one of claim 2-4 in the preparation of Chlamydia psittaci detection reagent or test kit. 10. the application of the detection reagent described in claim 5 or 6 in the preparation of Chlamydia psittaci detection reagent or test kit.

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