Primer group and application thereof

Abstract

The invention discloses a primer group and application thereof. The primer group comprises a first round of primer group and a second round of primer group, the first round of primer group is a species-specific multiplex amplification PCR primer group designed according to species of microorganisms to be detected, and the second round of primer group comprises a nested PCR primer group designed by utilizing a multiplex amplification region and a universal probe in an Amplifluor system. By utilizing the primer group provided by the invention, high-sensitivity, high-specificity and low-cost detection on target microorganisms can be realized.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a primer set and application thereof. Background Art

[0002] Polymerase Chain Reaction (PCR) was first proposed by Kary Mullis in 1983. It is a molecular biology technique that simulates the natural replication process of DNA in vitro to amplify specific DNA fragments. Real-time PCR (RT-PCR), also known as quantitative PCR (qPCR), combines the amplification ability of PCR with fluorescence detection technology. It can monitor the process of DNA amplification in real time, thereby achieving quantitative analysis of target genes. It is widely used in gene expression research, clinical diagnosis, forensic identification and food testing. According to different chemical principles, qPCR can be divided into the following two categories. One is the fluorescent dye method, which uses fluorescent dyes that can be inserted into double-stranded DNA, such as SYBR Green I and EvaGreen. These dyes will emit fluorescent signals after binding to DNA, and the fluorescence intensity is proportional to the amount of DNA. This method is simple and low-cost, but it may produce background signals due to non-specific amplification. The other is the fluorescent probe method, which uses specific fluorescent probes to detect PCR products. According to the different probe types, it can be divided into the following subtypes. There are three common methods: The first is the primer-probe method, such as Scorpions and etc. These probes are used as primers in the PCR amplification process to form specific signals; the second is the hydrolysis probe method, such as TaqMan probes. These probes are hydrolyzed by DNA polymerase during PCR amplification to release fluorescent signals. This is one of the most commonly used probe types; the third is the hybridization probe method, such as molecular beacons. These probes bind to the target DNA under specific conditions and emit fluorescent signals.

[0003] At present, the qPCR method based on the TaqMan probe method is widely used in the field of microbial detection. Compared with the fluorescent dye method, this method has higher specificity and can detect various microorganisms efficiently and accurately, but this method also has some limitations. For example, the cost of probe synthesis is high. Since TaqMan probes need to be specifically designed and synthesized according to different target microbial sequences, the cost is relatively high. In addition, although the TaqMan probe method has high specificity and sensitivity, in some cases, such as for the detection of certain complex samples or low copy number target microorganisms, its detection sensitivity is limited, and additional pretreatment steps or optimization of experimental conditions may be required to improve the detection effect. Summary of the invention

[0004] In view of this, in order to make up for the deficiencies of the prior art, the present invention is proposed. The object of the present invention is to provide a primer set and its application. The primer set includes a first-round primer set and a second-round primer set. The first-round primer set is a species-specific multiplex amplification PCR primer set designed according to the microbial species to be detected, and the second-round primer set includes a nested PCR primer set designed using the multiplex amplification region and a universal probe in the Amplifluor system. The primer set provided by the present invention can achieve high sensitivity, high specificity and low-cost detection of target microorganisms.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a method for designing primers, the method comprising: designing a first-round species-specific multiplex amplification PCR primer set for the microbial species to be detected, designing a nested PCR primer set using the multiplex amplification region, and making the nested PCR primer set and the universal probe in the Amplifluor system together form a second-round primer set.

[0007] In the present invention, primer refers to a short nucleic acid molecule. It can anneal with a complementary target nucleic acid molecule through nucleic acid hybridization to form a hybrid between the primer and the target nucleic acid chain. The primer can be extended along the target nucleic acid molecule by a polymerase. Therefore, the primer can be used to amplify the target nucleic acid molecule, wherein the sequence of the primer is specific to the target nucleic acid molecule.

[0008] In the present invention, the primer set consists of at least two pairs of primer pairs. The primer pair includes at least one forward primer and at least one reverse primer, wherein the primers are specific for the amplification of microbial nucleic acid molecules. The forward or upstream primer is a primer in the 5' direction relative to a reference point on the nucleic acid sequence; the reverse or downstream primer is a primer in the 3' direction relative to a reference point on the nucleic acid sequence. Generally speaking, at least one forward and one reverse primer are included in the amplification reaction.

[0009] Furthermore, the universal probe comprises a reporter group and a quencher group.

[0010] Furthermore, the universal probe has a hairpin structure.

[0011] Furthermore, one end of the hairpin structure is a reporter group, and the other end is a quencher group.

[0012] Furthermore, the reporter group includes FAM, FITC, HEX, VIC, TET, JOE, ROX, CY3, CY5 or CY7.

[0013] Furthermore, the quenching group includes BHQ1, BHQ2, BHQ3 or DABCYL.

[0014] Furthermore, the reporter group is at the 5' end.

[0015] Furthermore, the quenching group is at the 3' end.

[0016] Furthermore, the reporter group is selected from FAM, HEX, and VIC.

[0017] Furthermore, the quenching group is selected from BHQ1.

[0018] Furthermore, the sequence of the universal probe includes the nucleotide sequences shown in SEQ ID NO:37 and SEQ ID NO:38.

[0019] In the present invention, the source of the sample of the microorganism to be detected is not particularly limited. The sample of the microorganism to be detected can be derived from, but is not limited to, cells, tissues, body fluids, such as skin, mucous membranes, blood, blood derivatives, extracted bile, living tissue, tears, milk, dandruff, surface cleaning fluid, urine, sputum, cerebrospinal fluid, prostatic fluid, pus, bone marrow aspirate, middle ear effluent, bronchoalveolar lavage, tracheal aspirate, nasopharyngeal aspirate, oropharyngeal aspirate or saliva. The sample of the microorganism to be detected can also be derived from non-living substances such as food, soil, air, and water.

[0020] Further, the sample of the microorganism to be tested can be derived from humans or non-human animals. Non-human animals include non-human vertebrates, more preferably mammals, and mammals refer to all members of the class Mammalia, such as domesticated livestock (e.g., cattle, horses, pigs), pets (e.g., dogs, cats), or rodents.

[0021] Furthermore, the primers provided by the present invention can be used for the detection of multiple species of microorganisms at the same time.

[0022] Furthermore, the number of the microbial species to be detected is at least two.

[0023] Furthermore, the number of microbial species to be tested is four.

[0024] In the present invention, the species of the microorganism is not limited, and the microorganism includes bacteria, viruses or fungi.

[0025] Furthermore, the microorganism is selected from bacteria.

[0026] Furthermore, the bacteria include Gram-positive bacteria or Gram-negative bacteria.

[0027] Furthermore, the Gram-positive bacteria include Staphylococcus, Streptococcus or Enterococcus.

[0028] Furthermore, the Staphylococcus genus includes Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus.

[0029] Furthermore, the Gram-negative bacteria include Enterobacteriaceae, Vibrioaceae, and Acinetobacter.

[0030] Furthermore, the Enterobacteriaceae family includes Escherichia coli, Klebsiella pneumoniae, Enterobacter, Citrobacter, and Proteus.

[0031] Furthermore, the genus Acinetobacter includes calcoacetic acid Acinetobacter, luffy Acinetobacter, baumannii, hemolytic Acinetobacter, johnsonii, and johnsonii.

[0032] Furthermore, the bacteria is selected from Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae.

[0033] Furthermore, the method also includes screening of primer sets.

[0034] Further, the method comprises:

[0035] (1) Select the core genes of the microbial species to be tested and design the first round of primers;

[0036] (2) Design nested PCR primers using the multiple amplification regions.

[0037] Furthermore, primer sets can be designed using primer design software or manually designed.

[0038] Furthermore, the primers are 1-3 pairs of primers with the smallest penalty in the primer design software.

[0039] Furthermore, the first round of primer amplification length is 170bp-250bp.

[0040] Furthermore, the length of the first round primers ranges from 18 bp to 23 bp.

[0041] Furthermore, the first round primer annealing temperature ranges from 57°C to 63°C.

[0042] Furthermore, the GC content of the first round primers ranges from 40% to 60%.

[0043] Furthermore, the method also includes a first-round primer specificity evaluation method, the steps of which include: using the NCBI nt library as a reference database, if an amplified sequence or primer is only uniquely aligned to its source species, it is considered to be species-specific.

[0044] Furthermore, the nested PCR primer amplification length is 45bp-150bp.

[0045] Furthermore, the nested PCR primers have a length ranging from 18 bp to 23 bp.

[0046] Furthermore, the nested PCR primer annealing temperature ranges from 57°C to 63°C.

[0047] Furthermore, the GC content of the nested PCR primers ranges from 40% to 60%.

[0048] Furthermore, the method also includes a nested PCR primer specificity evaluation method, the steps of which include: using the first round amplification sequence of the species to be detected as a reference database, if an amplification sequence or primer is only uniquely aligned to the first round amplification sequence of its source species, it is considered to be specific.

[0049] In a specific embodiment of the present invention, the primer design method comprises:

[0050] (1) Select the core genes in each species, design primers using primer3, select the primer set that meets the filtering criteria, and then evaluate the species specificity of the amplified sequences and their primers, retain the amplified sequences and their primers with species specificity, and select 1-3 pairs of primers and their amplified sequences with the smallest primer3 penalty as the input of step 2. The filtering criteria are: amplification length is 170bp-250bp; primer length range is 18bp-23bp; annealing temperature range is 57℃-63℃; GC content range is 40%-60%. The specificity evaluation method is: using the NCBI nt library as the reference database, if an amplified sequence or primer is only uniquely aligned to its source species, it is considered to be species specific;

[0051] (2) Design primers for nested PCR using the multiple amplification regions in (1). The design method is as follows: select the amplified sequence of the first round of amplification of each species, use primer3 to design primers, and select 1-3 pairs of primers that meet the filtering criteria and have the smallest primer3 penalty as the primer pairs to be tested. The filtering criteria are: amplification length of 45bp-150bp; primer length range of 18bp-23bp; annealing temperature range of 57℃-63℃; GC content range of 40%-60%; specificity within the first round of target amplification range, that is, the first round of amplification sequences of all species to be tested are used as the reference database. If a certain amplification sequence or primer is only uniquely aligned to the first round of amplification sequence of its source species, it is considered to be specific.

[0052] A second aspect of the present invention provides a primer set for detecting microorganisms, wherein the primer set includes a first-round primer set and a second-round primer set.

[0053] Furthermore, the second round primer set includes nested PCR primers designed using multiple amplification regions amplified by the first round primers.

[0054] Furthermore, the second round primer set is composed of nested PCR primers designed using the multiple amplification regions amplified by the first round primers and a universal probe in the Amplifluor system.

[0055] Further, the first round primer set includes a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the primer pairs shown in SEQ ID NOs: 1 and 2, the primer pair shown in SEQ ID NOs: 3 and 4, the primer pair shown in SEQ ID NOs: 5 and 6, the primer pair shown in SEQ ID NOs: 7 and 8, the primer pair shown in SEQ ID NOs: 9 and 10, the primer pair shown in SEQ ID NOs: 11 and 12, the primer pair shown in SEQ ID NOs: 13 and 14, the primer pair shown in SEQ ID NOs: 15 and 16, and the primer pair shown in SEQ ID NOs: 17 and 18.

[0056] In the present invention, identity refers to the identity between two or more nucleic acid sequences, expressed in terms of identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences. Homologs or orthologs of nucleic acid sequences have relatively high levels of sequence identity when aligned using standard methods. The primers disclosed herein are not limited to the exact sequences shown, and as will be appreciated by those skilled in the art, the sequences may be altered, if desired, without significantly affecting the ability of the primers to function.

[0057] Further, the first round primer set includes any one of the primer pairs shown in SEQ ID NOs: 1 and 2, the primer pairs shown in SEQ ID NOs: 3 and 4, the primer pairs shown in SEQ ID NOs: 5 and 6, the primer pairs shown in SEQ ID NOs: 7 and 8, the primer pairs shown in SEQ ID NOs: 9 and 10, the primer pairs shown in SEQ ID NOs: 11 and 12, the primer pairs shown in SEQ ID NOs: 13 and 14, the primer pairs shown in SEQ ID NOs: 15 and 16, and the primer pairs shown in SEQ ID NOs: 17 and 18.

[0058] Further, the first round primer set is selected from the primer pair shown in SEQ ID NOs: 1 and 2, the primer pair shown in SEQ ID NOs: 5 and 6, the primer pair shown in SEQ ID NOs: 7 and 8, and the primer pair shown in SEQ ID NOs: 13 and 14.

[0059] Further, the nested PCR primer set includes a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the primer pairs shown in SEQ ID NOs: 19 and 20, the primer pair shown in SEQ ID NOs: 21 and 22, the primer pair shown in SEQ ID NOs: 23 and 24, the primer pair shown in SEQ ID NOs: 25 and 26, the primer pair shown in SEQ ID NOs: 27 and 28, the primer pair shown in SEQ ID NOs: 29 and 30, the primer pair shown in SEQ ID NOs: 31 and 32, the primer pair shown in SEQ ID NOs: 33 and 34, and the primer pair shown in SEQ ID NOs: 35 and 36.

[0060] Further, the nested PCR primer set includes any one of the primer pairs shown in SEQ ID NOs: 19 and 20, the primer pair shown in SEQ ID NOs: 21 and 22, the primer pair shown in SEQ ID NOs: 23 and 24, the primer pair shown in SEQ ID NOs: 25 and 26, the primer pair shown in SEQ ID NOs: 27 and 28, the primer pair shown in SEQ ID NOs: 29 and 30, the primer pair shown in SEQ ID NOs: 31 and 32, the primer pair shown in SEQ ID NOs: 33 and 34, and the primer pair shown in SEQ ID NOs: 35 and 36.

[0061] Furthermore, the nested PCR primer set is selected from the primer pair shown in SEQ ID NOs: 19 and 20, the primer pair shown in SEQ ID NOs: 23 and 24, the primer pair shown in SEQ ID NOs: 25 and 26, and the primer pair shown in SEQ ID NOs: 31 and 32.

[0062] Furthermore, the universal probe comprises a reporter group and a quencher group.

[0063] Furthermore, the universal probe has a hairpin structure.

[0064] Furthermore, one end of the hairpin structure is a reporter group, and the other end is a quencher group.

[0065] Furthermore, the reporter group includes FAM, FITC, HEX, VIC, TET, JOE, ROX, CY3, CY5 or CY7.

[0066] Furthermore, the quenching group includes BHQ1, BHQ2, BHQ3 or DABCYL.

[0067] Furthermore, the reporter group is at the 5' end.

[0068] Furthermore, the quenching group is at the 3' end.

[0069] Furthermore, the reporter group is selected from FAM, HEX, and VIC.

[0070] Furthermore, the quenching group is selected from BHQ1.

[0071] Furthermore, the sequence of the universal probe includes the nucleotide sequences shown in SEQ ID NO:37 and SEQ ID NO:38.

[0072] In the present invention, the primer set may further include:

[0073] (1) a nucleic acid sequence complementary to the primer set described above;

[0074] (2) A nucleic acid sequence that hybridizes with the nucleic acid sequence described in (1) under stringent conditions.

[0075] In the present invention, complementary nucleic acid sequences are formed by two complementary strands of double-stranded DNA or RNA chains consisting of base pairs. When the bases of one nucleic acid molecule form hydrogen bonds with the bases of another nucleic acid molecule, complementary binding occurs. In general, the base adenine (A) is complementary to thymine (T) and uracil (U), while cytosine (C) is complementary to guanine (G).

[0076] In the present invention, nucleic acid molecules can complement each other even when all bases of each molecule do not fully form hydrogen bonds. For example, hybridization with complementary nucleic acid sequences can occur under conditions of different stringencies, wherein the complementary strand will bind at some but not all nucleotide sites. In the present invention, stringent conditions refer to conditions that form specific hybridization and do not form non-specific hybridization.

[0077] In the present invention, hybridization means that two completely or partially complementary nucleic acid sequences are combined in a parallel or antiparallel direction under specific hybridization test conditions, thereby forming the ability of a stable structure with a double-stranded region. The two component chains of this double-stranded structure (sometimes referred to as hybrid chains) are combined together by hydrogen bonds. Although these hydrogen bonds are most commonly formed between nucleotides containing the bases adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G) on a single nucleic acid chain, base pairing can also be formed between bases that are not members of these "classical" pairs. Non-canonical base pairing is well known in the art.

[0078] Furthermore, the microorganism includes Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae.

[0079] Furthermore, at least one of the two primers in the primer pair is labeled with a labeling substance.

[0080] Furthermore, the labeling substance may be directly bound to the primer or may be bound via a linker.

[0081] The linker may be any linker commonly used in the art, and specifically, for example, a nucleic acid of 1 to 3 bases is preferred, a DNA of 1 to 3 bases is more preferred, a DNA of 2 bases is further preferred, and the 2 bases of adenine (A)-adenine (A) are particularly preferred.

[0082] Furthermore, the labeling substance includes fluorescent dyes, chemiluminescent compounds, radioactive isotopes, enzymes, and colored particle biotin.

[0083] Furthermore, the fluorescent dye includes fluorescent organic dyes, fluorescent quantum dots, fluorescent dyads, fluorescent carbon dots, graphene quantum dots or other carbon-based fluorescent nanostructures, fluorescent proteins, and nanostructures based on fluorescent DNA origami.

[0084] Furthermore, the chemiluminescent compounds include luminol, acridinium ester, terpyridine ruthenium, lucigenin, lophanine, and peroxy oxalate esters.

[0085] Furthermore, the radioisotope comprises 3 H. 123 I. 125 I. 131 I. 18 F. 11 C. 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 32 P. 33 P. 35 S.

[0086] Furthermore, the enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase.

[0087] Furthermore, the colored particles include gold, silver and platinum particles.

[0088] In the present invention, "fluorescent dye", "fluorophore", "fluorochrome", and "dye" are used interchangeably to refer to fluorescent chemical compounds or structures, and may be particularly one of the following: fluorescent organic dyes, fluorescent quantum dots, fluorescent dyads, fluorescent carbon dots, graphene quantum dots or other carbon-based fluorescent nanostructures, fluorescent proteins, and fluorescent DNA origami-based nanostructures. Among organic fluorescent dyes, the term "fluorescent dye" refers in particular to derivatives of xanthenes (e.g. fluorescein, rhodamine, Oregon Green, Texas), cyanines (e.g. cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine), derivatives, squarenine rotaxane derivatives, naphthalene, coumarin, oxadiazoles, anthracenes (anthraquinone, DRAQ5, DRAQ7, CyTRAK Orange), pyrene (Cascade Blue), oxazines (Nile Red, Nile Blue, Cresyl Violet, Oxazine 170), acridines (proflavine, acridine orange, acridine yellow), arylmethines (auramine, crystal violet, malachite green), tetrapyrroles (porphine, phthalocyanine, bilirubin), dipyrromethene (BODIPY, aza-BODIPY), phosphorescent dyes or luminescent dyes.

[0089] In the group of fluorescent proteins, in particular members of the green fluorescent protein (GFP) family, including but not limited to GFP and GFP-like proteins (e.g. DsRed, TagRFP) and their (monomeric) derivatives (e.g. EBFP, ECFP, EYFP, Cerμlaen, mTurquoise2, YFP, EYFP, mCitrine, Venus, YPet, Superfolder GFP, mCherry, mPlum).

[0090] The third aspect of the present invention provides a product for detecting microorganisms, wherein the product comprises the primer set described in the second aspect of the present invention.

[0091] Furthermore, the products include chips, nucleic acid membrane strips, and test kits.

[0092] Furthermore, the kit also includes enzymes for PCR, dNTPs, buffer solution, Mg 2+ at least one reactant.

[0093] Furthermore, Mg 2+ Sources, including but not limited to MgCl 2 MgSO 4 .

[0094] Furthermore, the enzyme used for PCR includes DNA polymerase and / or RNA polymerase.

[0095] Furthermore, the DNA polymerase includes but is not limited to Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4 DNA polymerase, and Klenow fragment.

[0096] Furthermore, the buffer includes but is not limited to polymerase buffer and Tris hydrochloric acid buffer.

[0097] Furthermore, the kit also includes instructions.

[0098] Furthermore, the kit may contain bacterial genomic DNA used as a positive control for PCR and sterile water used as a negative control.

[0099] In the present invention, a suitable amount of primer is provided in a container, or is fixed on a matrix, and the primer can be provided to be suspended in an aqueous solution, or for example as a freeze-dried or lyophilized powder. The container in which the nucleic acid is provided can be any conventional container that can hold the form provided, for example a microcentrifuge tube, an ampoule or a bottle. The test kit can include labeled or unlabeled probes for microbial nucleic acid sequences.

[0100] In some applications, primers can be provided in pre-measured single-use amounts in separate, typically disposable tubes or equivalent containers. Using such an arrangement, a sample to be tested for the presence of a microorganism can be added to a separate tube and amplified directly.

[0101] The amount of nucleic acid primers provided in the kit can be any suitable amount and can depend on the target market for the product. For example, if the kit is suitable for research or clinical applications, the amount of each nucleic acid primer provided can be an amount sufficient to initiate several PCR amplification reactions. The kit can contain more than two primers to facilitate PCR amplification of a larger number of microbial nucleic acid sequences.

[0102] Furthermore, the microorganism includes Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae.

[0103] The fourth aspect of the present invention provides a method for detecting microorganisms, the method comprising amplifying using the primer set described in the second aspect of the present invention, or amplifying using the product described in the third aspect of the present invention.

[0104] Further, the method comprises:

[0105] (1) performing a first round of amplification using a first round primer set;

[0106] (2) Amplification and detection using the second round of primer sets.

[0107] Furthermore, the method can detect at least a single copy sample of the microorganism to be detected.

[0108] Furthermore, the method can achieve stable detection of samples with at least 5 copies of the microorganism to be detected.

[0109] Furthermore, the microorganism includes Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae.

[0110] The fifth aspect of the present invention provides use of the primer set described in the second aspect of the present invention in the preparation of a product for detecting diseases caused by microorganisms.

[0111] Furthermore, the microorganism includes Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae.

[0112] The sixth aspect of the present invention provides use of the primer set described in the second aspect of the present invention or the product described in the third aspect of the present invention in detecting microorganisms.

[0113] Furthermore, the microorganism includes Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae.

[0114] A seventh aspect of the present invention provides a primer design system, the system comprising: a first-round primer design unit and a second-round primer design unit.

[0115] Furthermore, the first round primer design unit can design first round species-specific multiplex amplification PCR primers for the microbial species to be detected, and the second round primer design unit can design second round nested PCR primers using the multiplex amplification region.

[0116] Furthermore, the primer design system also includes a detection unit.

[0117] Furthermore, the detection unit can use the primers designed by the first round primer design unit for amplification, and use the primers designed by the second round primer design unit and the universal probe in the Amplifluor system for further amplification and detection.

[0118] Furthermore, the microorganism includes Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae.

[0119] The present invention has the following advantages and beneficial effects:

[0120] The present invention provides a primer set and its application. The primer set includes a first round primer set and a second round primer set. The first round primer set is a species-specific multiple amplification PCR primer set designed according to the microbial species to be detected, and the second round primer set includes a nested PCR primer set designed using the multiple amplification region and a universal probe in the Amplifluor system. The primer set provided by the present invention can achieve high sensitivity, high specificity and low cost detection of target microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Figure 1 This is a schematic diagram of the amplification principle.

[0122] Figure 2 These are the amplification curves of the MNAP process and Taqman-qPCR process for Escherichia coli and Acinetobacter baumannii. Figure 2A is the amplification curve of the MNAP process for Escherichia coli; Figure 2B is the amplification curve of the Taqman-qPCR process for Escherichia coli; Figure 2C is the amplification curve of the MNAP process for Acinetobacter baumannii; and Figure 2D is the amplification curve of the Taqman-qPCR process for Acinetobacter baumannii. DETAILED DESCRIPTION

[0123] The present invention is further described below in conjunction with specific examples, which are only used to explain the present invention and are not to be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purposes of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer.

[0124] Example 1: Development of a fluorescence PCR detection method integrating multiple nested PCR and Amplifluor technology (Multiplex Nested Amplifluor PCR, MNAP) process

[0125] 1. Experimental Materials

[0126] Samples: Acinetobacter baumannii (Aba), Staphylococcus aureus (Sau), Escherichia coli (Eco), and Klebsiella pneumoniae (Kpn).

[0127] Reagents: Microbial genome extraction kit, Amplifluor amplification system, Taqman amplification system, PCRmix, AMPure XP magnetic beads, etc.

[0128] 2. Experimental methods:

[0129] Step 1: Design the first round of species-specific multiplex amplification PCR primers for four microorganisms, Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae. The design method is as follows: select the core genes in each species, design primers using primer3, select the primer set that meets the following filtering criteria, and then evaluate the species specificity of the amplified sequences and their primers, retain the amplified sequences and their primers with species specificity, and select 1-3 pairs of primers and their amplified sequences with the smallest primer3 penalty as input for step 2. The filtering criteria are: amplification length of 170bp-250bp; primer length range of 18bp-23bp; annealing temperature range of 57℃-63℃; GC content range of 40%-60%. The specificity evaluation method is: using the NCBI nt library as the reference database, if an amplified sequence or primer is only uniquely aligned to its source species, it is considered to be species-specific.

[0130] The primer sequences are shown in Table 1 below:

[0131] Table 1 Sequences of first round primer sets

[0132]

[0133]

[0134] Step 2: Use the multiple amplification regions in step 1 to design primers for the second round of nested PCR. The design method is as follows: select the amplified sequence of the first round of amplification of each species, use primer3 to design primers, and select 1-3 pairs of primers that meet the following filtering criteria and have the smallest primer3 penalty as the primer pairs to be tested. The filtering criteria are: amplification length of 45bp-150bp; primer length range of 18bp-23bp; annealing temperature range of 57℃-63℃; GC content range of 40%-60%; specificity within the first round of target amplification range, that is, the first round of amplification sequences of all species to be tested are used as the reference database. If a certain amplification sequence or primer is only uniquely aligned to the first round of amplification sequence of its source species, it is considered to be specific.

[0135] The primer sequences are shown in Table 2 below:

[0136] Table 2 Second round primer set sequences

[0137]

[0138]

[0139] Step 3: Use the multiplex amplification PCR primer set designed in step 1 that is specific to the microbial species to be detected to perform the first round of amplification on the nucleic acid of the sample to be detected.

[0140] The amplification system is shown in Table 3:

[0141] Table 3 Amplification system

[0142] Element 40μl reaction system PCRMix 20μl Multiplex Amplification Primer Set 4μl Nucleic acid to be tested 1μl Nuclease-free water 15μl Total volume 40μl

[0143] The amplification conditions are shown in Table 4:

[0144] Table 4 Amplification conditions

[0145]

[0146] Step 4: Purify the PCR product using magnetic beads.

[0147] After the reaction, the PCR product was purified by magnetic beads. The purification steps are as follows: Take 40μl AMPure XP magnetic beads and place them in the PCR product. Mix thoroughly and let it stand for 5 minutes. Place the PCR tube in a magnetic rack to separate the magnetic beads and liquid. After the solution is clear, carefully remove the supernatant. Keep the PCR tube in the magnetic rack, add 200μl of 80% ethanol freshly prepared with nuclease-free water to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant. Repeat the rinse once. Keep the PCR tube in the magnetic rack and open the lid to dry the magnetic beads at room temperature. Add 22μl nuclease-free water, pipette until fully mixed, and let it stand at room temperature for 5 minutes. Centrifuge the PCR tube briefly and place it in a magnetic rack to stand. After the solution is clear, carefully transfer 20μl of supernatant to a new PCR tube. Use Qubit to measure the concentration of the recovered product.

[0148] Step 5: Use the nested PCR primers designed in step 2 to perform Amplifluor detection using ABI7500 as the detection instrument.

[0149] Figure 1 This is a schematic diagram of the amplification and detection principles of steps 3-5.

[0150] The amplification system is shown in Table 5:

[0151] Table 5 Amplification system

[0152] Element 20 μl reaction system AmplifluorMix 10μl Nested amplification primer pairs 2μl Nucleic acid to be tested 1μl Nuclease-free water 7μl Total volume 20μl

[0153] The amplification conditions are shown in Table 6:

[0154] Table 6 Amplification conditions

[0155]

[0156] Step 6: Analyze the amplification curve and Ct value to determine whether the target is successfully detected.

[0157] Step 7: Extraction and testing of nucleic acid of the strain to be tested.

[0158] The nucleic acids of each strain of Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae were extracted and 10 copies / rxn of simulated samples were prepared to verify and screen the primer combinations designed in steps 1-2.

[0159] 3. Experimental results

[0160] This experiment used 10 copies / rxn simulated samples to verify and screen the primer combinations obtained in step 1-2. The test results are shown in Table 7 below:

[0161] Table 7 Test results

[0162]

[0163] According to the above test results, the primer pairs with the smallest Ct value (i.e., the most sensitive detection) for each species to be detected (Aba-mF1 / Aba-mR1+Aba-nF1 / Aba-nR1, Sau-mF / Sau-mR+Sau-nF / Sau-nR, Eco-mF1 / Eco-mR1+Eco-nF1 / Eco-nR1 and Kpn-mF1 / Kpn-mR1+Kpn-nF1 / Kpn-nR1) were selected as the final primer combination for performance testing to evaluate the minimum detection limit and specificity of MNAP.

[0164] Example 2: Highly sensitive detection of Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae

[0165] 1. Experimental Materials

[0166] Samples: Acinetobacter baumannii (Aba), Staphylococcus aureus (Sau), Escherichia coli (Eco), and Klebsiella pneumoniae (Kpn).

[0167] Reagents: Microbial genome extraction kit, Amplifluor amplification system, Taqman amplification system, PCRmix, AMPure XP magnetic beads, etc.

[0168] 2. Experimental methods:

[0169] Using the quantitative strains of the four species, simulated samples with concentrations of 100, 10, 5, 1 and 0.5 copies / ml for each strain of Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae were prepared by dilution in proportion, and then nucleic acid was extracted and tested for this series of samples.

[0170] 3. Experimental results

[0171] The results are shown in Table 8:

[0172] Table 8 Sensitivity test results

[0173]

[0174]

[0175] This experiment used simulated samples of different concentrations to test the MNAP process, and used the Taqman-qPCR method as a control. It can be seen from the table that the MNAP method of the present invention has a higher detection sensitivity, and can detect a single copy of Bauman, Staphylococcus aureus, Escherichia coli and Pneumococcus at the lowest, and can achieve stable detection of 5 copies / ml of Bauman, Escherichia coli and Pneumococcus samples, which is 20 times higher than the conventional Taqman probe method. It is worth noting that Figure 2 From the amplification curves (taking the amplification curves of Bowman's and large intestine as examples; the curves from the upper left corner to the lower right corner are 100, 10, 5, 1 copies / ml and NC samples respectively), it can be seen that MNAP has a more complete and clear amplification curve, which helps to accurately determine the presence or absence of the target.

[0176] Example 3: Specificity test of MNAP

[0177] 1. Experimental Materials

[0178] Samples: Closely related strains of Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae were selected for specificity testing, see Table 9 for details.

[0179] Table 9 Species used for specific detection

[0180]

[0181]

[0182] Reagents: Microbial genome extraction kit, Amplifluor amplification system, Taqman amplification system, PCRmix, AMPure XP magnetic beads, etc.

[0183] 2. Experimental methods:

[0184] The experimental steps are the same as those in Example 1, except for the test samples.

[0185] 3. Experimental results

[0186] The results are shown in Table 10. It can be seen from the table that no amplification and Ct value detection were found for the closely related strains, indicating that the method has good detection specificity for Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae, and there is no cross-reaction between closely related strains.

[0187] Table 10 Specificity test results

[0188]

[0189] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for designing a primer, characterized in that: The method comprises: designing a first-round species-specific multiplex amplification PCR primer set for the microbial species to be detected, designing a nested PCR primer set using the multiplex amplification region, and making the nested PCR primer set and the universal probe in the Amplifluor system together form a second-round primer set; Preferably, the universal probe comprises a reporter group and a quencher group; Preferably, the reporter group comprises FAM, FITC, HEX, VIC, TET, JOE, ROX, CY3, CY5 or CY7; Preferably, the quenching group comprises BHQ1, BHQ2, BHQ3 or DABCYL; Preferably, the sequence of the universal probe includes the nucleotide sequence shown in SEQ ID NO:37 and SEQ ID NO:38; Preferably, the number of microbial species to be detected is at least two; Preferably, the number of microbial species to be detected is four; Preferably, the microorganism comprises bacteria, virus or fungus; Preferably, the microorganism is selected from bacteria; Preferably, the bacteria include Gram-positive bacteria or Gram-negative bacteria; Preferably, the Gram-positive bacteria include Staphylococcus, Streptococcus or Enterococcus; Preferably, the Staphylococcus genus includes Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus; Preferably, the Gram-negative bacteria include Enterobacteriaceae, Vibrioaceae, and Acinetobacter; Preferably, the Enterobacteriaceae family includes Escherichia coli, Klebsiella pneumoniae, Enterobacter, Citrobacter, and Proteus; Preferably, the genus Acinetobacter includes Acinetobacter calcoaceticus, Acinetobacter luffii, Acinetobacter baumannii, Acinetobacter haemolyticus, Acinetobacter junii, and Acinetobacter johnsonii; Preferably, the bacteria is selected from Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae.

2. The method according to claim 1, characterized in that The method comprises screening of primer sets; Preferably, the method comprises: (1) Select the core genes of the microbial species to be tested and design the first round of primers; (2) Designing nested PCR primers using multiple amplified regions; Preferably, the primer set can be designed using primer design software or manually designed; Preferably, the primers are 1-3 pairs of primers with the smallest penalty in the primer design software; Preferably, the first round of primer amplification length is 170bp-250bp; Preferably, the first round primers are in the range of 18 bp to 23 bp in length; Preferably, the first round primer annealing temperature ranges from 57°C to 63°C; Preferably, the GC content of the first round primers ranges from 40% to 60%; Preferably, the method further comprises a first round of primer specificity evaluation method, the steps of which include: using the NCBI nt library as a reference database, if a certain amplified sequence or primer is only uniquely aligned to its source species, it is considered to be species specific; Preferably, the nested PCR primer amplification length is 45bp-150bp; Preferably, the nested PCR primers range in length from 18 bp to 23 bp; Preferably, the nested PCR primer annealing temperature ranges from 57°C to 63°C; Preferably, the GC content of the nested PCR primers ranges from 40% to 60%; Preferably, the method also includes a nested PCR primer specificity evaluation method, the steps of which include: using the first round amplification sequence of the species to be detected as a reference database, if an amplification sequence or primer is only uniquely aligned to the first round amplification sequence of its source species, it is considered to be specific.

3. A primer set for detecting microorganisms, characterized in that: The primer set includes a first round primer set and a second round primer set; Preferably, the first round primer set is a multiplex amplification PCR primer specific to the microbial species to be detected; Preferably, the second round primer set includes nested PCR primers designed using multiple amplification regions amplified by the first round primers; Preferably, the second round primer set is composed of nested PCR primers designed using the multiple amplification regions amplified by the first round primers and a universal probe in the Amplifluor system; Preferably, the first round primer set comprises a sequence having at least 90% identity with any one of the primer pairs shown in SEQ ID NOs: 1 and 2, the primer pair shown in SEQ ID NOs: 3 and 4, the primer pair shown in SEQ ID NOs: 5 and 6, the primer pair shown in SEQ ID NOs: 7 and 8, the primer pair shown in SEQ ID NOs: 9 and 10, the primer pair shown in SEQ ID NOs: 11 and 12, the primer pair shown in SEQ ID NOs: 13 and 14, the primer pair shown in SEQ ID NOs: 15 and 16, and the primer pair shown in SEQ ID NOs: 17 and 18; Preferably, the first round primer set includes any one of the primer pair shown in SEQ ID NOs: 1 and 2, the primer pair shown in SEQ ID NOs: 3 and 4, the primer pair shown in SEQ ID NOs: 5 and 6, the primer pair shown in SEQ ID NOs: 7 and 8, the primer pair shown in SEQ ID NOs: 9 and 10, the primer pair shown in SEQ ID NOs: 11 and 12, the primer pair shown in SEQ ID NOs: 13 and 14, the primer pair shown in SEQ ID NOs: 15 and 16, and the primer pair shown in SEQ ID NOs: 17 and 18; Preferably, the first round primer set is selected from the primer pair shown in SEQ ID NOs: 1 and 2, the primer pair shown in SEQ ID NOs: 5 and 6, the primer pair shown in SEQ ID NOs: 7 and 8, and the primer pair shown in SEQ ID NOs: 13 and 14; Preferably, the nested PCR primer set comprises a sequence having at least 90% identity with any one of the primer pairs shown in SEQ ID NOs: 19 and 20, the primer pair shown in SEQ ID NOs: 21 and 22, the primer pair shown in SEQ ID NOs: 23 and 24, the primer pair shown in SEQ ID NOs: 25 and 26, the primer pair shown in SEQ ID NOs: 27 and 28, the primer pair shown in SEQ ID NOs: 29 and 30, the primer pair shown in SEQ ID NOs: 31 and 32, the primer pair shown in SEQ ID NOs: 33 and 34, and the primer pair shown in SEQ ID NOs: 35 and 36; Preferably, the nested PCR primer set includes any one of the primer pair shown in SEQ ID NOs: 19 and 20, the primer pair shown in SEQ ID NOs: 21 and 22, the primer pair shown in SEQ ID NOs: 23 and 24, the primer pair shown in SEQ ID NOs: 25 and 26, the primer pair shown in SEQ ID NOs: 27 and 28, the primer pair shown in SEQ ID NOs: 29 and 30, the primer pair shown in SEQ ID NOs: 31 and 32, the primer pair shown in SEQ ID NOs: 33 and 34, and the primer pair shown in SEQ ID NOs: 35 and 36; Preferably, the nested PCR primer set is selected from the primer pair shown in SEQ ID NOs: 19 and 20, the primer pair shown in SEQ ID NOs: 23 and 24, the primer pair shown in SEQ ID NOs: 25 and 26, and the primer pair shown in SEQ ID NOs: 31 and 32; Preferably, the universal probe comprises a reporter group and a quencher group; Preferably, the reporter group comprises FAM, FITC, HEX, VIC, TET, JOE, ROX, CY3, CY5 or CY7; Preferably, the quenching group comprises BHQ1, BHQ2, BHQ3 or DABCYL; Preferably, the sequence of the universal probe includes the nucleotide sequence shown in SEQ ID NO:37 and SEQ ID NO:38; Preferably, the microorganism comprises Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae.

4. The primer set according to claim 3, characterized in that: At least one of the two primers in the primer pair is labeled with a labeling substance; Preferably, the labeling substance includes fluorescent dyes, chemiluminescent compounds, radioactive isotopes, enzymes, colored particles biotin; Preferably, the fluorescent dye comprises fluorescent organic dye, fluorescent quantum dots, fluorescent dyads, fluorescent carbon dots, graphene quantum dots or other carbon-based fluorescent nanostructures, fluorescent proteins, and fluorescent DNA origami-based nanostructures; Preferably, the chemiluminescent compound includes luminol, acridinium ester, terpyridine ruthenium, lucigenin, lophanine, and peroxy oxalate esters; Preferably, the radioisotope comprises 3 H. 123 I. 125 I. 131 I. 18 F. 11 C. 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 32 P. 33 P. 35 S; Preferably, the enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, acetylcholinesterase; Preferably, the colored particles include gold, silver, or platinum particles.

5. A product for detecting microorganisms, characterized in that: The product comprises the primer set according to any one of claims 3-4; Preferably, the product includes a chip, a nucleic acid membrane strip, and a test kit; Preferably, the kit also includes enzymes for PCR, dNTPs, buffer solution, Mg 2+ at least one reactant; Preferably, the enzyme used for PCR comprises DNA polymerase and / or RNA polymerase; Preferably, the buffer comprises polymerase buffer and Tris hydrochloric acid buffer; Preferably, the kit further comprises instructions; Preferably, the microorganism comprises Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae.

6. A method for detecting microorganisms, characterized in that: The method comprises amplifying using the primer set described in any one of claims 3-4, or amplifying using the product described in claim 5.

7. The method according to claim 6, characterized in that The method comprises: (1) performing a first round of amplification using a first round primer set; (2) using the second round of primer sets for amplification and detection; Preferably, the method can detect at least a single copy sample of the microorganism to be detected; Preferably, the method can achieve stable detection of at least 5 copies of the sample of the microorganism to be detected; Preferably, the microorganism comprises Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae.

8. Use of the primer set according to any one of claims 3 to 4 in the preparation of a product for detecting diseases caused by microorganisms; Preferably, the microorganism comprises Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae.

9. Use of the primer set according to any one of claims 3 to 4 or the product according to claim 5 in detecting microorganisms; Preferably, the microorganism comprises Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae.

10. A primer design system, characterized in that: The system comprises: a first round primer design unit, a second round primer design unit; Preferably, the first round primer design unit can design first round species-specific multiplex amplification PCR primers for the microbial species to be detected, and the second round primer design unit can design second round nested PCR primers using the multiplex amplification regions; Preferably, the microorganism comprises Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae.

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