CN111154916A - Primer group, detection reagent and kit for respiratory tract pathogen multiple RPA detection - Google Patents

Primer group, detection reagent and kit for respiratory tract pathogen multiple RPA detection Download PDF

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CN111154916A
CN111154916A CN202010074966.5A CN202010074966A CN111154916A CN 111154916 A CN111154916 A CN 111154916A CN 202010074966 A CN202010074966 A CN 202010074966A CN 111154916 A CN111154916 A CN 111154916A
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biotin
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陈愉生
李鸿茹
孙益乐
许能銮
郑奎城
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FUJIAN CENTER FOR DISEASE CONTROL AND PREVENTION
Shanghai Tellgen Life Science Co ltd
FUJIAN PROVINCIAL HOSPITAL
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Abstract

The invention provides a primer group for detecting multiple RPA of respiratory pathogens, a detection reagent and a kit, wherein the primer group for detecting the multiple RPA of high specificity is successfully obtained by designing and modifying at least 5 respiratory pathogens of human cytomegalovirus, adenovirus, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, respiratory syncytial virus, staphylococcus aureus, streptococcus pneumoniae, mycoplasma pneumoniae, chlamydia pneumoniae, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, stenotrophomonas maltophilia and legionella pneumophila, and the primer group is adopted to prepare the detection reagent and manufacture the kit, so that the RPA of the pathogenic bacteria of the respiratory tract of at most 15 reoccurrence can be detected, the time required by the whole detection process can be shortened, and the steps are simplified.

Description

Primer group, detection reagent and kit for respiratory tract pathogen multiple RPA detection
Technical Field
The invention relates to the field of molecular biology, in particular to a primer group, a detection reagent and a kit for detecting multiple RPA of respiratory tract pathogens.
Background
The RPA technique (collectively referred to as "recombinase-mediated amplification technique") is known as a nucleic acid detection technique that can replace the PCR technique. The RPA technique is a technique that enables rapid amplification of nucleic acids at a constant temperature. RPA technology relies primarily on three enzymes: a recombinase capable of binding a primer, a single-stranded DNA binding protein (SSB), and a DNA polymerase; the optimum reaction temperature for the mixture of these three enzymes is around 37 ℃. The principle of the RPA technology is as follows: the recombinase binds to the primer to form a protein-DNA complex which searches for homologous sequences in the double-stranded DNA, and once the primer searches for a homologous sequence which is completely complementary to the homologous sequence, the double-stranded DNA template is unzipped, the primer is paired and bound to the homologous sequence with the help of the single-stranded DNA binding protein, DNA synthesis is initiated under the action of DNA polymerase, and the target region on the double-stranded DNA template is exponentially amplified, and at the same time, the replaced DNA strand is bound to SSB to prevent further replacement. In amplification systems of the RPA technique, one synthesis event is initiated by two opposing primers. The whole process is carried out very quickly, and detectable levels of amplification product are usually obtained within 1 h. Compared with the PCR technology, the whole reaction of the RPA technology does not need high-temperature circulation, is simple and quick, and is particularly suitable for being used in a non-laboratory detection place with a large number of samples.
The key to the RPA technology is the design of primers. Existing PCR amplification primers are mostly unsuitable because the RPA primers are longer than the PCR primers, typically requiring up to 30-38 bases. Furthermore, in designing RPA primers, denaturation temperature is no longer a critical factor affecting the amplification primers. Primer design for RPA is not as mature as traditional PCR, which requires the investigator to do the primer design and optimization himself. Because the RPA technology is usually carried out under isothermal condition, the mutual interference among multiple amplification products caused by the difference of Tm values and the amplification efficiencies among different respiratory tract pathogenic primer groups in multiple detection cannot be avoided by adjusting the annealing temperature and the cooling rate in the reaction like common PCR amplification, and the difficulty of obtaining the high-specificity RPA amplification primer suitable for multiple detection is very high. At present, the single detection by using the RPA technology is reported more, and at most 4 detections are realized.
The clinical diagnosis of acute respiratory infectious diseases is complex, and the pathogeny causing the same symptom is often more than 10. Based on the advantages of simple operation and rapid amplification of the RPA technology. The development of RPA primer groups, detection reagents and kits for multiple detection of respiratory tract pathogens is urgently hoped for in clinical medicine.
Disclosure of Invention
An object of the present invention is to provide a primer set for multiplex RPA detection of respiratory pathogens, which can detect 5 or more (up to 15) respiratory pathogens at a time by amplifying RPA in any one of DNA and RNA in a sample to be detected and detecting the same, and which can shorten the time required for the entire process and simplify the steps.
The RPA primer group for detecting the respiratory tract pathogen multiple RPA comprises at least 5 pairs of 15 pairs of primers, wherein the nucleotide sequences of the 15 pairs of primers are respectively as follows:
① human cytomegalovirus (HCMV for short)
HCMV-F：5'biotin–CGGTACGGGCTGCAGGTAAAGTGCGATCAAGAACG 3'、
HCMV-R：5'NH2-C6-TTTTTTTTTCGTGCTTTTTAGCCTCTTTTTTTTTTTGCAGCTGCG
TCACGGGTCTAGCCGGCCATAATCAC 3'；
② adenovirus (ADV for short)
ADV-F：5'biotin –CTCGGAGTACCTGAGCCCCGGGCTGGTGCAGTTCG 3'、
ADV-R：5'NH2-C6-TTTTTTTTTTCGTCGTGCGTAGGCGCTTTTTTTTTTACCGTGGGGTT
TCTAAACTTGTTATTCAGGCTGA 3'；
③ parainfluenza virus type 1 (PIV 1 for short)
PIV1-F：5'biotin –AAATGAGACTACAGATTACTCGAGTGAAGGTATA 3'、
PIV1-R：5'NH2-C6-TTTTTTTTTTTAGAAAAAGGATGGTCATTTTTTTTTAAGTTATATCT
TCATTTTTGTATCGATGAGATT 3'；
④ parainfluenza virus type 2 (PIV 2 for short)
PIV2-F：5'biotin –CAACTGATCTAGCTGAACTGAGACTTGCTTTCTA 3'、
PIV2-R：5'NH2-C6-TTTTTTTTTTCAACTGCAGGATTGATTTTTTTTTTTTTGGCCCAT
TGCCCTGTTGTATTTGGAAGAGATAT 3'；
⑤ parainfluenza virus type 3 (PIV 3 for short)
PIV3-F1：5'biotin –ACAGATAGGGATAATAACTGTAAACTCAGACT 3'、
PIV3-R：5'NH2-C6-TTTTTTTTTTAGTGCTAGAGAACATGTTTTTTTTTTCTTCCTATTGT
CATTTATGTTAAAAGTATGAGA 3'；
⑥ respiratory syncytial virus (RSV for short)
RSV-F：5'biotin –TCCTCACTTCTCCAGTGTAGTATTAGGCAATGCT 3'、
RSV-R：5'NH2-C6-TTTTTTTTTTAATCACACCATTTTCTTTTTTTTTTTTTGAGTTGTTCA
GCATATGCCTTTGCTGCATCATATA 3'；
⑦ Staphylococcus Aureus (SA)
SA-F：5'biotin –GAAAAAATCTTAGAATTAATGGAAGCTGTAGATACTT 3'、
SA-R：5'NH2-C6-TTTTTTTTTCAACAGTACCACGACCAGTTTTTTTTTTTGAATACGTC
CTCAACTGGCATCATGAATGGTTTGT 3'；
⑧ Streptococcus Pneumoniae (SP)
SP-F：biotin – CGTGCTCCGATGACTTATAGTATTGATTTGCCTGGTT 3'、
SP-R：5'NH2-C6-TTTTTTTTTTCCAACAAATCGTTTACCTTTTTTTTTTTCCGCGAACA
CTTGAATTGCTGGGGTCTTCCA 3'；
⑨ Mycoplasma pneumoniae (MP for short)
MP-F：5'biotin– GCGCTAAGGGCATCACTGCCGGCAGTGGCAGTCAAC 3'、
MP-R：5'NH2-C6-TTTTTTTTTCGAGGTCGTATAAGGCTTTTTTTTTTTCGCCCGGCGAG
GTCATACCGGCGTAACGCAAAGGTGG 3'；
⑩ Mycoplasma pneumoniae (MP for short)
CP-F：5'biotin–GCCATTTTATCTCCAACTTGAAGTTTTCTCTTAGA 3'、
CP-R：5'NH2-C6-TTTTTTTTTTTATTTCCGTGTCGTCCTTTTTTTTTTTGCCATTTTATCT
CCAACTTGAAGTTTTCTCTTAGA 3'；
⑾ Klebsiella pneumoniae (KP for short)
KP-F：5'biotin–CTGGATCTGACCCTGCAGTACCAGGGTAAAAAC 3'、
KP-R：5'NH2-C6-TTTTTTTTTGCTCGCTGATTATCATGTTTTTTTTTTTGCGGTTGGCC
GATCATGTTTGGCCTGATCCTCTCTGCG 3'；
⑿ Acinetobacter baumannii (AB for short)
AB-F：5'biotin–AGCTCAAGTCCAATACGACGAGCTAAATCTTGAT 3'、
AB-R：5'NH2-C6-TTTTTTTTTATTTAAGTGGGATGGTTTTTTTTTTTTTATTCCCAGAATG
GGAAAAGGACATGACCCTAGGC 3'；
⒀ Pseudomonas aeruginosa (abbreviation: PA)
PA-F：5'biotin–CTTCAACGAGAACCTGCTCTGCTTCACCAACAAC、
PA-R：5'NH2-C6-TTTTTTTTTTCGGCCTCGATGTAGTTGTTTTTTTTTTTCAGGTTACG
CGTCAGCGCCGAACGGAAACCGGCC 3'；
⒁ Stenotrophomonas Maltophilia (SM)
SM-F：5'biotin–AGCGACGAGCGAACGCGGACTAGCCCTTAAGCTGAT 3'、
SM-R：5'NH2-C6-TTTTTTTTTTCTCGTCTTCACTGAAATGTTTTTTTTTTTCCCTTTCA
GATACAGGGCTATCACCTTCTATGGCCAC 3'；
⒂ Legionella pneumophila (LP for short)
LP-F：5'biotin–GCAATGGCTGCAACCGATGCCACATCATTAGCT 3'
LP-R：5'NH2-C6-TTTTTTTTTTACATCTATGCCTTGATTTTTTTTTTTCCCCAAATCGG
CACCAATGCTATAAGACAACTTA 3'。
The "-F" in the HCMV-F, AD-F, PIV1-F, PIV2-F, PIV3-F, RSV-F, SA-F, SP-F, MP-F, CP-F, KP-F, AB-F, PA-F, SM-F, LP-F refers to the upstream primer modified by biotin at the 5 'end of the corresponding pathogen, and the "-R" in the HCMV-R, AD-R, PIV1-R, PIV2-R, PIV3-R, RSV-R, SA-R, SP-R, MP-R, CP-R, KP-R, AB-R, PA-R, SM-R, LP-R refers to the downstream primer modified by amino at the 5' end of the corresponding pathogen.
Compared with the multiplex PCR detection method, because the multiplex RPA technology is carried out under the constant temperature condition, the mutual interference among a plurality of amplification products caused by the difference of Tm values and the amplification efficiencies among different respiratory tract pathogen primer groups causes greater difficulty in obtaining the high-specificity primers suitable for the multiplex RPA detection, and therefore, the sensitivity and the specificity of the multiplex RPA detection method are generally poorer than those of the multiplex PCR detection method, so that the multiplex RPA detection method disclosed by the prior art can only achieve 4 detections at most, and as the number of the primer pairs in the primer group increases, the mutual interference among each pair of primers, among various amplification products and among the primers and the products can be realized, the detection sensitivity can be greatly reduced, and the accuracy of the detection result can be reduced. According to the invention, through numerous experiments and matching experiences, a conserved sequence on a pathogen genome is selected, the conserved sequence is used as an amplification sequence, primer design is carried out according to a base complementary pairing principle, meanwhile, when a downstream primer is prepared, a plurality of corresponding bases (namely ' anchor sequences ') complementary with an amplification fragment are added to the 5 ' end of the downstream primer obtained by primary screening, and meanwhile, partial bases on the primer are modified, so that the binding efficiency of the primer and a template is increased, the detection sensitivity can be greatly improved even if the pathogen of respiratory diseases is detected by 15 times, the specificity is not reduced, meanwhile, the time required by the whole flow is greatly shortened, and the operation steps are simplified.
The second purpose of the invention is to provide a respiratory tract pathogen multiple RPA detection reagent, which contains all the upstream primers in the primer group, primer-microsphere conjugates, free four kinds of deoxymononucleotides (dNTPs), enzyme, single-strand binding protein, phycoerythrin (SA-PE), buffer solution and ATP, wherein the primer-microsphere conjugates comprise at least 5 kinds of primer-microsphere conjugates formed by coupling each downstream primer in the primer group with microspheres with different detection markers in a one-to-one correspondence manner, and the enzyme mainly comprises recombinase and DNA polymerase. The invention directly adopts the downstream primer and the microsphere for coupling preparation of the primer-microsphere coupling body, and adds the primer-microsphere coupling body into an amplification system for amplification, the process of anchoring the labeled primer to the microsphere is completed in the amplification process, compared with the existing PCR detection method (the existing PCR detection method generally needs to firstly carry out PCR amplification, then carries out hybridization on the prepared probe-microsphere coupling body and an amplification product, needs to open a reaction tube after hybridization, manually adds phycoerythrin into the hybridization product of the reaction tube, incubates for 15 minutes at the temperature of 37 ℃ for binding reaction, and can carry out liquid suspension chip system detection after the binding reaction), the invention can reduce the binding process of the hybridization process and the phycoerythrin, shorten the detection time, simplify the steps and also reduce the manual participation.
The invention also aims to provide a using method of the respiratory tract pathogen multiple RPA detection reagent, which comprises the following steps: any one of RNA and DNA in the extracted sample to be detected is taken as a nucleic acid template, and is completely mixed with the respiratory tract pathogen multiple RPA detection reagent at one time, then RPA amplification is carried out, and finally a liquid suspension chip system is adopted to detect the amplification product. Just because the applicant develops a primer group with high specificity, all the upstream primers and the primer-microsphere conjugates can be mixed together for reaction and detection at one time, and the using method is simpler and quicker.
In the specific implementation process, the reaction temperature is 37-42 ℃ during RPA amplification, and the reaction time is 10-20 min.
The fourth purpose of the invention is to provide a kit containing the respiratory tract pathogen multiple RPA detection reagent.
Detailed Description
Embodiments of the invention will now be described in detail:
the experimental procedures used in the following examples and comparative examples are conventional ones unless otherwise specified.
Materials, reagents and the like used in the following examples and comparative examples are commercially available unless otherwise specified.
Examples
The primer group for detecting the respiratory tract pathogen multiple RPA comprises 15 pairs of primers, and the construction process of the primers comprises the following steps:
1) the inventor uses Clustal Omega and Vector NTI Suite 8.0 software to compare the DNA sequence of known specific genes of pathogens (also called as pathogens) and screens out the specific sequence of the acute respiratory infectious pathogens, and the nucleotide sequence is shown as follows:
① specific sequence of cytomegalovirus (HCMV for short):
AAGTTTGTGCCCCAACGGTACGGGCTGCAGGTAAAGTGCGATCAAGAACGCGATAACGCCGATCACAAACAGCGTGACGATGACCTGCCATCGACGGTGATTATGGCCGGCTAGACCCGTGACGCAGCTGCAGAGGCTAAAAAGCACGC；
② adenovirus (ADV for short):
CGCAGTGGTCTTACATGCACATCTCGGGCCAGGACGCCTCGGAGTACCTGAGCCCCGGGCTGGTGCAGTTCG
CCCGCGCCACCGAGACGTACTTCAGCCTGAATAACAAGTTTAGAAACCCCACGGTGGCGCCTACGCACGACGTGACCACA；
③ specific sequence of human parainfluenza virus type 1 (PIV 1):
CCGGAAATTTCTCATACCTATGACATCAACGACAACAGGAAATCATGTTCTGTAATAGCTGCAGGAACAAGGGGTTATCAGTTATGCTCCTTGCCCACTGTAAATGAGACTACAGATTACTCGAGTGAAGGTATAGAAGATTTAGTATTTGACATATTAGATCTCAAGGGAAAGACCAAATCTCATCGATACAAAAATGAAGATATAACTTTTGACCATCCTTTTTCTGCAATGTATCCAAGTGTAGGAAGTGGGATAAAGATTGAAAATACACTCATCTTCTTAGGGTACGGTGGCTTAACAACTCCGCTCAAGG；
④ specific sequence of human parainfluenza virus type 2 (PIV 2):
ATGGAATCAATCGCAAAAGCTGTTCAGTCACTGCTATACCAGGAGGTTGTGTCTTGTATTGCTATGTAGCTA
CAAGATCTGAGAAAGAAGATTATGCCACAACTGATCTAGCTGAACTGAGACTTGCTTTCTATTATTATAATGATACCTTTATTGAAAGAGTCATATCTCTTCCAAATACAACAGGGCAATGGGCCACAATCAATCCTGCAGTTGGAAGCGGGAT
CTATCATC ；
⑤ specific sequence of human parainfluenza virus type 3 (PIV 3):
CTCGAGGTTGTCAGGATATAGGAAAATCATATCAAGTCTTACAGATAGGGATAATAACTGTAAACTCAGACTTGGTACCTGACTTAAATCCCAGGATCTCTCATACTTTTAACATAAATGACAATAGGAAGTCATGTTCTCTAGCACTCCTAAATACAGATGTATATCAACTGTGTTCAACTCCCAAAG ；
⑥ specific sequence of respiratory syncytial virus (RSV for short):
CAAGTTGTTGAGGTTTATGAATATGCCCAAAAATTGGGTGGTGAAGCAGGATTCTACCATATATTGAACAAC
CCAAAAGCATCATTATTATCTTTGACTCAATTTCCTCACTTCTCCAGTGTAGTATTAGGCAATGCTGCTGGCCTAGGCATAATGGGAGAGTACAGAGGTACACCGAGGAATCAAGATCTATATGATGCAGCAAAGGCATATGCTGAACAACTCAAAGAAAATGGTGTGATTAACTACAGTGTACTAGACTTGACAGCAGAA ；
⑦ specific sequence of Staphylococcus aureus (SA for short):
GGAAGTTCGTGACTTATTAAGCGAATATGACTTCCCAGGTGACGATGTACCTGTAATCGCTGGTTCAGCAT
TAAAAGCTTTAGAAGGCGATGCTCAATACGAAGAAAAAATCTTAGAATTAATGGAAGCTGTAGATACTTACATTCCAACTCCAGAACGTGATTCTGACAAACCATTCATGATGCCAGTTGAGGACGTATTCTCAATCACTGGTCGTGGTACTGTTGCTACAGGCCGTGTTGAACGTGGTCAAATCAAAGTTGGTGAAGAAGTTGAAATCATCGGTTTACATGACACATCTAAAACAACTGU ；
⑧ specific sequence of Streptococcus pneumoniae (SP for short):
GTGATATTTCTGTAACAGCTACCAACGACAGTCGCCTCTATCCTGGAGCACTTCTCGTAGTGGATGAGACCT
TGTTAGAGAATAATCCCACTCTTCTTGCGGTCGATCGTGCTCCGATGACTTATAGTATTGATTTGCCTGGTTTGGCAAGTAGCGATAGCTTTCTCCAAGTGGAAGACCCCAGCAATTCAAGTGTTCGCGGAGCGGTAAACGATTTGTTGGCTAAGTGGCATCAAGATTATGGTCAGGTCAATAATGTCCCAGCTAGAATGCAGTATGAAAAAATCACGGCTCACAGCATGGAACAACTCAAGGTCAAGTTTGGTTCTGACTTTGAAAAGACAGGGAATTCTC ；
⑨ specific sequence of Mycoplasma pneumoniae (MP for short):
TGCCATCTACCCGCGCTTAACCCCGTGAACGTATCGTAACACGAGCTTTTCCTCCCTCCCCCTCACGGGTGAAAATCCCGGGGCGTGGGCCTTAGTGCGCGACAACAGCGCTAAGGGCATCACTGCCGGCAGTGGCAGTCAACAAACCACGTATGAACCCACCCGAACCGAAGCGGCTTTGACCGCATCAACCACCTTTGCGTTACGCCGGTATGACCTCGCCGGGCGCGCCTTATACGACCTCGATTTTTCGAAGTTAAACCCGCAAACGCCCACGCGCGACCAAACCGGGCAGATCAC；
⑩ specific sequence of Chlamydia pneumoniae (CP for short):
TTTATTTCCGTGTCGTCCAGCCATTTTATCTCCAACTTGAAGTTTTCTCTTAGAGGCAACGTAGACTTTAACTTGGCGAATGACACCATGATCTAAATCTGCATCTCCCTCACGAATATGCTCAACT ；
⑾ specific sequence of Klebsiella pneumoniae (KP for short):
CTGGATCTGACCCTGCAGTACCAGGGTAAAAACGAAGGCCGTGAAGCGAAGAAACAGAACGGCGACGG ；
⑿ specific sequence of Acinetobacter baumannii (AB for short):
AACCAACACGCTTCACTTCCTTAGACATGAGCTCAAGTCCAATACGACGAGCTAAATCTTGATAAACTGGAA
TAGCGGAAGCTTTCATGGCATCGCCTAGGGTCATGTCCTTTTCCCATTCTGGGAATAACCTTTTTTTACCATCCCACTTAAATACTTCTGTGGTGGTTGCCTTATGGTGCTCAAGGCCGATCAAAGCATTAAGCATTTTGAAGGTCGAAGCAGGTACATACTCGGTCGAAGCACGA ；
⒀ specific sequence of Pseudomonas aeruginosa (PA for short):
GGCGTGGGTGTGGAAGTCGCCTTGCAGTGGAACGACAGCTTCAACGAGAACCTGCTCTGCTTCACCAACAAC
ATCCCGCAGCGTGACGGCGGTACCCACCTGGCCGGTTTCCGTTCGGCGCTGACGCGTAACCTGAACAACTACATCGAGGCCGAAGGCCTGGCGAAGAAGTTCAAGATCGCCAC ；
⒁ specific sequence of stenotrophomonas maltophilia (abbreviated as SM):
CAGCCTGCGAAAAGTATCGGGGAGCTGGCAACAAGCTTTGATCCGGTAATGTCCGAATGGGGAAACCCACCC
GCTTGCGGGTATCCTGCAGTGAATACATAGCTGCTGGAAGCGAACCTGGTGAACTGAAATATCTAAGTAACCAGAGGAAAAGAAATCAACCGAGATTCCGTAAGTAGCGACGAGCGAACGCGGACTAGCCCTTAAGCTGATTTGGTTCTAGGAAAACACTCTGGAAAGAGTGGCCATAGAAGGTGATAGCCCTGTATCTGAAAGGGCCATTTCAGTGAAGACGAGTAGGGCGGGGCACGTGAAACCCTGTCTGAACATGGGGGGACCATCCTCCAAGGCTAAATACTACTGACCGACCGATAGTGAACCAGTACCGTGAGGGAAAGGCGAAAAGAACCCCGGAGAGGGGAGTGAAATAGAACCTGAAACCGTGTGCGTACAAGCAGTAGGAGCTCCGCAAGGAGTGACTGCGTACCTTTTGTATAATGGGTCAGCGACTTACTGTTCGTGGCAAGCTTAA ；
⒂ specific sequence of legionella pneumophila (LP for short):
TGGGGCTTGCAATGTCAACAGCAATGGCTGCAACCGATGCCACATCATTAGCTACAGACAAGGATAAGTTGTCTTATAGCATTGGTGCCGATTTGGGGAAGAATTTTAAAAATCAAGGCATAGATGTTAATCCGGAAGCAATGGCTAAAGGCATGCAAGACGCTATGAGTGGCGCTCAATTGGCTTTAACCGAACAGC。
2) according to the specific sequences, bioinformatics software is applied, and a large amount of screening and transformation design are carried out, so that the primer group for detecting the multiple RPA is finally obtained, wherein the primers are shown in Table 1.
TABLE 1 primer group for multiplex RPA detection of the present application
Figure 878735DEST_PATH_IMAGE001
The "-F" of HCMV-F, AD-F, PIV1-F, PIV2-F, PIV3-F, RSV-F, SA-F, SP-F, MP-F, CP-F, KP-F, AB-F, PA-F, SM-F, LP-F in Table 1 refers to the upstream primer modified by biotin at the 5 'end of the corresponding pathogen, while the "-R" of HCMV-R, AD-R, PIV1-R, PIV2-R, PIV3-R, RSV-R, SA-R, SP-R, MP-R, CP-R, KP-R, AB-R, PA-R, SM-R, LP-R refers to the downstream primer modified by amino at the 5' end of the corresponding pathogen.
And (II) the respiratory tract pathogen multiple RPA detection reagent contains all upstream primers, primer-microsphere conjugates, free four kinds of deoxymononucleotides (dNTP), enzyme, single-strand binding protein, phycoerythrin (SA-PE), buffer solution and ATP in the primer group, wherein the enzyme mainly comprises recombinase and DNA polymerase, and when the reagent is used, DNA in a sample to be detected is extracted as a nucleic acid template and is mixed with all detection reagents at one time, and then RPA amplification is carried out. Wherein the primer-microsphere conjugate comprises 15 pathogenic primer-microsphere conjugates, and the preparation method of the primer-microsphere conjugate of each pathogen comprises the following steps: respectively mixing 100nmol of downstream primer with 1ng of microspheres with different colors and 2- (N-morpholinyl) ethanesulfonic acid solution with the concentration of 25mM and the pH value of 6.0, then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride for reaction for 2h, then sealing with bovine serum albumin solution, and finally washing with Tween-20 solution to obtain a primer-microsphere coupling body; the color of the microspheres coupled and combined with different pathogenic downstream primers is different. The obtained primer-microsphere conjugates of 15 pathogens are respectively: HCMV-R microsphere 1, ADV-R microsphere 2, PIV1-R microsphere 3, PIV2-R microsphere 4, PIV3-R microsphere 5, RSV-R microsphere 6, SA-R microsphere 7, SP-R microsphere 8, MP-R microsphere 9, CP-R microsphere 10, KP-R microsphere 11, AB-R microsphere 12, PA-R microsphere 13, SM-R microsphere 14 and LP-R microsphere 15.
When in use, the primer group and the multiple RPA detection reagent prepared by the primer group are adopted to establish an RPA amplification system, and the specific composition of the RPA amplification system is shown in Table 2:
TABLE 2
Figure 264717DEST_PATH_IMAGE002
Reaction conditions are as follows: and adding the reaction system into a PCR tube, putting the PCR tube into a Biometra T personal PCR instrument, setting the temperature to be 37-42 ℃ and setting the reaction time to be 10-20 min, and carrying out RPA amplification reaction.
(III) results of the detection
1) Detecting instruments and setting parameters: and (3) carrying out magnetic bead recognition and signal reading on the amplification product after the reaction by using a Luminex200 liquid chip scanner, and automatically interpreting the result by using software.
2) And (4) judging a result: microspheres with corresponding colors of certain pathogenic bacteria can be generated, and the microspheres are positive when the fluorescence value is more than or equal to 200; the fluorescence value is less than 150 and is negative; when the fluorescence value is between 150 and 200, the sample needs to be tested repeatedly, if the fluorescence value is larger than or equal to 150, the sample is judged to be positive, and if the fluorescence value is smaller than 150, the sample is judged to be negative.
3) Evaluation of sensitivity
The source of the sample DNA extract is shown in table 3 below:
TABLE 3
Figure 513295DEST_PATH_IMAGE003
10 samples of the 15 DNA/RNA extracts were provided0/μL、101/μL、102/μL、103/μL、104mu.L of 5 dilutions, the amount of sample DNA/RNA extract added during the detection was 1 uL. And 5 dilutions of pathogen DNA extract (i.e. DNA of a sample to be detected) are used as a DNA template, the kit is adopted for detection, the fluorescence value is read, and the sensitivity of detecting various pathogens by the acute respiratory infectious disease liquid chip detection method is determined. The fluorescence readings for each pathogen are now listed in table 4 below:
TABLE 4
Figure 174084DEST_PATH_IMAGE004
As can be seen from Table 4, the concentration of the pathogens of the acute respiratory infectious diseases is 103mu.L (i.e. 10)6mL) can detect the fluorescence value of more than or equal to 200 (namely the lowest detection limit is 106fu/ml). The lowest detection limit is lower than the lowest detection limit of pathogenic bacteria or conditioned pathogenic bacteria which are quantitatively cultured and separated by phlegm and published in the 7 th edition of internal medicine 2008 and can be identified as acute respiratory diseases: 107cfu/ml. Therefore, the invention has lower minimum detection limit and high sensitivity, and meets the requirement of detecting acute respiratory diseases.
Meanwhile, using artificially synthesized plasmids as nucleic acid templates (the nucleic acid templates comprise 15 plasmids including a plasmid carrying an HCMV amplification sequence, a plasmid carrying an AD amplification sequence, a plasmid carrying a PIV1 amplification sequence, a plasmid carrying a PIV2 amplification sequence, a plasmid carrying a PIV3 amplification sequence, a plasmid carrying an RSV amplification sequence, a plasmid carrying an SA amplification sequence, a plasmid carrying an SP amplification sequence, a plasmid carrying an MP amplification sequence, a plasmid carrying a CP amplification sequence, a plasmid carrying a KP amplification sequence, a plasmid carrying an AB amplification sequence, a plasmid carrying a PA amplification sequence and a plasmid carrying an SM amplification sequence), simulating samples to be detected, and performing primer verification according to the following processes: primer pairs of human cytomegalovirus, adenovirus, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, respiratory syncytial virus, staphylococcus aureus, streptococcus pneumoniae, mycoplasma pneumoniae, chlamydia pneumoniae, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, stenotrophomonas maltophilia and legionella pneumophila are combined with amplification fragments of 15 pathogens in a one-to-one correspondence manner, and 15-time amplification effects are verified (see table 9 below).
4) The specificity evaluation, also called as the true negative rate evaluation, namely the pathogen of non-acute respiratory infection is correctly judged as the pathogen percentage of non-current infection according to the standard of the detection method. Found in the detection of sensitivity (see table 5 below): since the detection is negative when the fluorescence value is below 150, which is not the pathogenic bacterium of this time, the specificity of the kit of the present application is 100%.
TABLE 5
Figure 191719DEST_PATH_IMAGE005
5) Good repeatability, when the concentration of pathogenic bacteria is 1 multiplied by 104The test was repeated three times for each pathogen at μ L, and the results are shown in table 6 below:
TABLE 6
Figure 483023DEST_PATH_IMAGE006
As can be seen from Table 6, the coefficient of variation is 0.05-0.11, and for synchronous detection of various pathogenic bacteria, the smaller the coefficient of variation, the better the repeatability.
5) And (3) simulating sample detection:
the applicant provides simulation specimens 1-4, wherein the simulation specimens 1-4 respectively comprise the following components (see table 7):
TABLE 7
Simulation specimen Containing pathogens
1 SA+AB+SP+SM+ADV+PIV3
2 KP+CP+SM+MP+RSV
3 SA+SP+SM+RSV+PIV2+HCMV
4 SA+AB+CP+SP+MP+ADV+PIV1
The detection results of the simulated samples 1-4 are shown in the following table 8:
TABLE 8
Figure 15635DEST_PATH_IMAGE007
In view of the fact that the types of pathogens in clinical samples are 5 types at most, the applicant provides a simulation sample containing 5-7 pathogens for detection, and as can be seen from tables 7 and 8, the invention can perform multiple synchronous detection on a positive sample containing more than 5 acute respiratory infectious disease pathogens, has high specificity of detection results and accurate detection results, and can completely meet the clinical detection requirements. On this basis, it can be further estimated that: the invention can also carry out multiplex synchronous detection on positive samples containing more than 7 acute respiratory infectious disease pathogens.
Comparative example
The comparative example differs from the above examples only in that: the base sequences of 15 pairs of primers of the primer group are shown in Table 9 below:
TABLE 9
Figure 417798DEST_PATH_IMAGE008
The primer sequence in the primer group of the invention is obtained by modifying the primer sequence of comparative example 1.
When the applicant uses the primer group of comparative example 1 to prepare the RPA detection reagent and the kit to carry out 15-fold detection verification on 15 artificially synthesized plasmids respectively carrying 15 pathogen amplification sequences, the following results are found: the detection sensitivity of the kit of comparative example 1 was very low. The applicant speculates that the reason may be: due to the steric hindrance of the primers on the microspheres, the binding rate of the primers and the templates is too low, so that the amplification efficiency of the primers is low, and the detection sensitivity is poor. Therefore, the applicant has specifically modified primers on microspheres to increase the binding efficiency of the primers to the template. To achieve this, the whole reaction system is optimized as follows: under the condition of keeping the upstream primer in the primer group unchanged, only the downstream primer in comparative example 1 is modified, and a plurality of corresponding bases complementary to the amplified fragment (namely, "anchor sequences", the underlined sequences in table 1 represent "anchor sequences") are added at the 5' end of the downstream primer, and meanwhile, partial bases on the downstream primer are modified (the bold bases in table 1 are modified bases).
The results of the 15-fold test verification for the comparative and example are shown in table 10 below:
TABLE 10 comparison of the test results of comparative example and example
Figure 352256DEST_PATH_IMAGE009
In order to increase the amplification effect of the primer on the surface of the microsphere without reducing the specificity of the primer, the applicant adds a sequence at the 5' end of the primer for increasing the binding efficiency of the primer and the template, and plays a role in capturing the template by an anchoring sequence; meanwhile, the 5 ' end base in the amplification primer is modified by Spacer 18 to limit the amplification of the ' anchor sequence ' and avoid the reduction of the specificity of the primer. By comparing the detection results of the comparative example 1 and the examples, it can be seen that the primer group of the present application effectively improves the amplification effect of RPA, thereby improving the detection sensitivity without causing the decrease of specificity.
Generally, the encoding techniques for microspheres suitable for liquid-state chip system detection mainly include methods such as fluorescent encoding and bar code etching. Thus, the microspheres of the present invention may be bar code etched microspheres or may be fluorescently encoded microspheres.
In addition, the RPA multiplex detection process of the present invention only requires 55min, while the existing PCR multiplex detection process requires more than 300min, as shown in Table 11 below. The primer group, the RPA detection reagent prepared by the primer group and the kit can obtain a detection result in advance for 4 hours on the basis of not reducing the detection performance.
TABLE 11
Figure 983437DEST_PATH_IMAGE010
As can be seen from the above table 11, the primer group, the RPA detection reagent and the kit prepared by using the primer group of the present invention have the most obvious change that the detection time is shortened; on the other hand, manual operation steps are reduced, the labor consumption can be reduced through the simplification of the manual operation steps, and human errors are reduced.
The above embodiments are merely preferred embodiments of the present invention, and should not be construed as limiting the invention. Modifications and substitutions based on the technical scheme of the invention can be made without departing from the spirit and the essence of the invention, and the invention also belongs to the protection scope of the invention.
Sequence listing
<110> Fujian province Hospital, Fujian province disease prevention and control center, Shanghai Jing Life science and technology corporation
<120> primer group for respiratory tract pathogen multiple RPA detection, detection reagent and kit
<130>DS-P19567
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Claims (5)

1. The RPA primer group for detecting the respiratory tract pathogen multiple RPA is characterized in that: the primer group consists of at least 5 pairs of 15 pairs of primers, and the nucleotide sequences of the 15 pairs of primers are respectively as follows:
human cytomegalovirus (HCMV for short)
HCMV-F：5'biotin–CGGTACGGGCTGCAGGTAAAGTGCGATCAAGAACG 3'、
HCMV-R：5'NH2-C6-TTTTTTTTTCGTGCTTTTTAGCCTCTTTTTTTTTTTGCAGCTGCG
TCACGGGTCTAGCCGGCCATAATCAC 3'；
② adenovirus (ADV for short)
ADV-F：5'biotin –CTCGGAGTACCTGAGCCCCGGGCTGGTGCAGTTCG 3'、
ADV-R：5'NH2-C6-TTTTTTTTTTCGTCGTGCGTAGGCGCTTTTTTTTTTACCGTGGGGTT
TCTAAACTTGTTATTCAGGCTGA 3'；
Parainfluenza virus type 1 (PIV 1 for short)
PIV1-F：5'biotin –AAATGAGACTACAGATTACTCGAGTGAAGGTATA 3'、
PIV1-R：5'NH2-C6-TTTTTTTTTTTAGAAAAAGGATGGTCATTTTTTTTTAAGTTATATCT
TCATTTTTGTATCGATGAGATT 3'；
Parainfluenza virus type 2 (PIV 2 for short)
PIV2-F：5'biotin –CAACTGATCTAGCTGAACTGAGACTTGCTTTCTA 3'、
PIV2-R：5'NH2-C6-TTTTTTTTTTCAACTGCAGGATTGATTTTTTTTTTTTTGGCCCAT
TGCCCTGTTGTATTTGGAAGAGATAT 3'；
⑤ parainfluenza virus type 3 (PIV 3 for short)
PIV3-F1：5'biotin –ACAGATAGGGATAATAACTGTAAACTCAGACT 3'、
PIV3-R：5'NH2-C6-TTTTTTTTTTAGTGCTAGAGAACATGTTTTTTTTTTCTTCCTATTGT
CATTTATGTTAAAAGTATGAGA 3'；
⑥ respiratory syncytial virus (RSV for short)
RSV-F：5'biotin –TCCTCACTTCTCCAGTGTAGTATTAGGCAATGCT 3'、
RSV-R：5'NH2-C6-TTTTTTTTTTAATCACACCATTTTCTTTTTTTTTTTTTGAGTTGTTCA
GCATATGCCTTTGCTGCATCATATA 3'；
⑦ Staphylococcus Aureus (SA)
SA-F：5'biotin –GAAAAAATCTTAGAATTAATGGAAGCTGTAGATACTT 3'、
SA-R：5'NH2-C6-TTTTTTTTTCAACAGTACCACGACCAGTTTTTTTTTTTGAATACGTC
CTCAACTGGCATCATGAATGGTTTGT 3'；
⑧ Streptococcus Pneumoniae (SP)
SP-F：biotin – CGTGCTCCGATGACTTATAGTATTGATTTGCCTGGTT 3'、
SP-R：5'NH2-C6-TTTTTTTTTTCCAACAAATCGTTTACCTTTTTTTTTTTCCGCGAACA
CTTGAATTGCTGGGGTCTTCCA 3'；
⑨ Mycoplasma pneumoniae (MP for short)
MP-F：5'biotin– GCGCTAAGGGCATCACTGCCGGCAGTGGCAGTCAAC 3'、
MP-R：5'NH2-C6-TTTTTTTTTCGAGGTCGTATAAGGCTTTTTTTTTTTCGCCCGGCGAG
GTCATACCGGCGTAACGCAAAGGTGG 3'；
⑩ Mycoplasma pneumoniae (MP for short)
CP-F：5'biotin–GCCATTTTATCTCCAACTTGAAGTTTTCTCTTAGA 3'、
CP-R：5'NH2-C6-TTTTTTTTTTTATTTCCGTGTCGTCCTTTTTTTTTTTGCCATTTTATCT
CCAACTTGAAGTTTTCTCTTAGA 3'；
⑾ Klebsiella pneumoniae (KP for short)
KP-F：5'biotin–CTGGATCTGACCCTGCAGTACCAGGGTAAAAAC 3'、
KP-R：5'NH2-C6-TTTTTTTTTGCTCGCTGATTATCATGTTTTTTTTTTTGCGGTTGGCC
GATCATGTTTGGCCTGATCCTCTCTGCG 3'；
⑿ Acinetobacter baumannii (AB for short)
AB-F：5'biotin–AGCTCAAGTCCAATACGACGAGCTAAATCTTGAT 3'、
AB-R：5'NH2-C6-TTTTTTTTTATTTAAGTGGGATGGTTTTTTTTTTTTTATTCCCAGAATG
GGAAAAGGACATGACCCTAGGC 3'；
⒀ Pseudomonas aeruginosa (abbreviation: PA)
PA-F：5'biotin–CTTCAACGAGAACCTGCTCTGCTTCACCAACAAC、
PA-R：5'NH2-C6-TTTTTTTTTTCGGCCTCGATGTAGTTGTTTTTTTTTTTCAGGTTACG
CGTCAGCGCCGAACGGAAACCGGCC 3'；
⒁ Stenotrophomonas Maltophilia (SM)
SM-F：5'biotin–AGCGACGAGCGAACGCGGACTAGCCCTTAAGCTGAT 3'、
SM-R：5'NH2-C6-TTTTTTTTTTCTCGTCTTCACTGAAATGTTTTTTTTTTTCCCTTTCA
GATACAGGGCTATCACCTTCTATGGCCAC 3'；
⒂ Legionella pneumophila (LP for short)
LP-F：5'biotin–GCAATGGCTGCAACCGATGCCACATCATTAGCT 3'
LP-R：5'NH2-C6-TTTTTTTTTTACATCTATGCCTTGATTTTTTTTTTTCCCCAAATCGG
CACCAATGCTATAAGACAACTTA 3'。
2. The reagent for multiplex RPA detection of respiratory pathogens, which comprises all the upstream primers in the RPA primer group for multiplex RPA detection of respiratory pathogens according to claim 1, primer-microsphere conjugates comprising at least 5 primer-microsphere conjugates formed by coupling each downstream primer in the RPA primer group for multiplex RPA detection of respiratory pathogens according to claim 1 with microspheres having different detection markers in a one-to-one correspondence manner, free four deoxymononucleotides (abbreviated as "dNTP"), an enzyme consisting essentially of a recombinase and a DNA polymerase, a single-strand binding protein, phycoerythrin (abbreviated as "SA-PE"), a buffer and ATP.
3. The use method of the respiratory tract pathogen multiplex RPA detection reagent comprises the following steps: any one of DNA and RNA in the extracted sample to be detected is taken as a nucleic acid template, and is mixed with the respiratory tract pathogen multiple RPA detection reagent according to claim 2 at one time, then RPA amplification is carried out, and finally, a liquid suspension chip system is adopted to detect the amplification product.
4. The method for using the multiple RPA detection reagent according to claim 3, wherein: the reaction temperature is 37-42 ℃ during RPA amplification, and the reaction time is 10-20 min.
5. A kit comprising the respiratory pathogenic multiplex RPA detection reagent according to claim 2.
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CN112626274A (en) * 2020-12-30 2021-04-09 济南国益生物科技有限公司 Primer probe set, kit and detection method for multiple detection of human parainfluenza virus subtype based on fluorescence RMA method
CN112831601A (en) * 2020-12-30 2021-05-25 济南国益生物科技有限公司 Primer probe set, kit and detection method for multiple detection of respiratory syncytial virus subtype based on fluorescent RMA method
NL2033870B1 (en) * 2022-09-01 2024-03-12 Baoding Childrens Hospital Kit for detection of mycoplasma pneumoniae, detection method and use thereof
CN117004771A (en) * 2023-06-14 2023-11-07 南京迪飞医疗器械有限公司 Rapid detection method and kit for respiratory virus multiplex detection nucleic acid

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