Primer probe combination product and kit for detecting brucella

Abstract

The present application relates to the technical field of molecular biology detection, and particularly relates to a primer probe combination product and kit for detecting Brucella. The present application realizes rapid determination of Brucella and accurate typing identification of B. abortus, B. melitensis and B. suis in the same detection process by constructing a multiplex primer probe combination containing genus level conservative targets and multiple species specific targets. The scheme reduces the operation complexity and time cost caused by traditional step-by-step detection, and improves the stability and reliability of the typing results through multi-target collaborative detection, and reduces the risk of misjudgment caused by single site variation. In addition, the preferred two-tube system and multi-channel detection mode are conducive to reducing the opening operation, reducing the cross contamination probability, and facilitating the standardization detection in different types of samples, and are conducive to improving the detection efficiency and result consistency.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, and in particular to a primer-probe combination product and kit for detecting Brucella. Background Technology

[0002] Brucellosis is a zoonotic infectious disease caused by bacteria of the genus Brucella. Current laboratory detection methods for Brucella mainly include bacterial isolation and culture, serological testing, and nucleic acid testing. Among these, nucleic acid amplification testing is currently the primary method for laboratory detection of brucellosis due to its rapid detection speed and high sensitivity. Bacterial isolation and culture, along with serological testing, are still the main methods used for confirmatory testing, but they have a long testing cycle and require specific experimental conditions. Serological methods are suitable for screening, but may have certain limitations in interpretation depending on the stage of infection and previous immunization / exposure.

[0003] Compared with the methods mentioned above, nucleic acid amplification detection has potential advantages such as a shorter detection cycle and the ability to obtain early etiological evidence, and has been gradually implemented in some laboratories and specific application scenarios in recent years. However, overall, the application of nucleic acid detection in brucellosis detection has not yet become widespread. One reason for this is that some existing nucleic acid detection protocols can only detect Brucella at the genus level, making it difficult to further distinguish between Brucella bovis, Brucella ovis, and Brucella swine in the same test; other typing protocols rely on a single typing site, which may lead to insufficient coverage or mistyping risks when dealing with strains from different regions or atypical strains. In addition, some detection procedures require separate genus detection and typing detection, which involves more steps, increases detection costs and the difficulty of contamination control, and is not conducive to promotion and application in grassroots or routine laboratories.

[0004] Therefore, there is an urgent need for a nucleic acid detection method, primer-probe combination, and kit that can simultaneously identify Brucella spp. and perform typing of Brucella bovis, Brucella ovis, and Brucella swine in a single test. This would improve detection efficiency, reduce operational complexity, and enhance the stability of typing results, thereby promoting the application of nucleic acid detection in relevant scenarios. Summary of the Invention

[0005] This invention covers the following technical solutions: One aspect of the present invention relates to a primer-probe combo product comprising a) to d): a) The primer pairs shown in SEQ ID NO: 1-2 and the probe shown in SEQ ID NO: 3; b) The primer pairs shown in SEQ ID NO: 4-5 and the probe shown in SEQ ID NO: 6; c) The primer pairs shown in SEQ ID NO: 7-8 and the probe shown in SEQ ID NO: 9; d) The primer pairs shown in SEQ ID NO: 10-11 and the probe shown in SEQ ID NO: 12.

[0006] Another aspect of the present invention relates to a kit containing a primer-probe combination product as described above.

[0007] Another aspect of the present invention relates to the use of the primer-probe combination product as described above in the preparation of a kit for differentiating infections of Brucella bovis, Brucella ovis, and Brucella swine.

[0008] This invention achieves rapid identification of Brucella genus and accurate typing of Brucella bovis, Brucella ovis, and Brucella suis in a single detection process by constructing a multiplex primer-probe combination containing genus-level conserved targets and multiple species-specific targets. This approach reduces the operational complexity and time cost of traditional step-by-step detection, while improving the stability and reliability of typing results through multi-target synergistic detection, reducing the risk of misclassification due to single-site variations. Furthermore, the optimized two-tube system and multi-channel detection method reduce the need for opening the tube, lower the probability of cross-contamination, and facilitate standardized detection in different sample types, thus improving detection efficiency and result consistency. Attached Figure Description

[0009] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0010] Figure 1 : A schematic diagram of the interpretation logic of one embodiment of the present invention.

[0011] Figure 2 Typical amplification curves of different targets in a two-tube multiplex fluorescent PCR system are shown below: A shows the typical amplification curve of the genotypic target IS711 in tube A; B shows the typical amplification curve of the bovine genotyping target alkB-IS711 in tube A; C shows the typical amplification curve of the ovine genotyping target BMEI1162-IS711 in tube B; D shows the typical amplification curve of the porcine genotyping target BS1330_II0657 in tube B.

[0012] Figure 3Representative amplification curves of the multiplex fluorescent PCR system at different concentration levels in this embodiment of the invention; A shows the amplification of IS711 target in tube A at different template concentrations; B shows the amplification of bovine typological target alkB-IS711 in tube A at different template concentrations; C shows the amplification of ovine typological target BMEI1162-IS711 in tube B at different template concentrations; D shows the amplification of porcine typological target BS1330_II0657 in tube B at different template concentrations.

[0013] Figure 4 : Schematic diagram of specificity / cross-reactivity detection in an embodiment of the present invention. Detailed Implementation

[0014] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0015] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) should be understood to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to constitute any limitation on the scope of protection of this invention. Unless the context clearly defines otherwise, the scientific and technical terms used herein, as well as terms and laboratory procedures in related fields such as protein and nucleic acid chemistry, molecular biology, and microbiology, are all conventional terms and standard methods well-known and widely used in the art. To facilitate understanding of the technical solutions of this invention, some related terms are further defined and explained below.

[0016] As used herein, the terms “and / or,” “or / and,” and “and / or” encompass any one of two or more of the relevant listed items, as well as any and all combinations of the relevant listed items, including any two of the relevant listed items, any more of the relevant listed items, or a combination of all the relevant listed items.

[0017] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0018] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0019] Furthermore, in describing representative embodiments of the invention, this specification may present the methods and / or processes of the invention as a specific sequence of steps. However, the method or process should not be limited to the specific order of the steps described herein, to the extent that the method or process does not depend on the specific order of the steps presented herein. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps presented in the specification should not be construed as a limitation of the claims. Additionally, the claims relating to the methods and / or processes of the invention should not be limited to the execution of their steps in the order they are written, and those skilled in the art will readily recognize that the sequence can be changed while still remaining within the spirit and scope of the invention.

[0020] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% can fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted.

[0021] As used in this invention, unless otherwise stated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0022] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity greater than or equal to 2.

[0023] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0024] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0025] In this invention, the term "brucellosis" refers to the genus Brucella (Brucella). Brucella These bacteria primarily infect animals such as cattle, dogs, pigs, sheep, and goats.

[0026] In this invention, the term "IS711" refers to Brucella bacteria, which are widely found in Brucella (IS711). BrucellaAn IS711 sequence is an insertion sequence in the genome, belonging to a type of mobile genetic element, which is usually distributed in multiple copies in different bacterial species and at different genomic locations. The IS711 sequence has relatively conserved nucleic acid sequence characteristics and can be used as a universal target for detection at the Brucella level.

[0027] In this invention, the term "alkB-IS711" refers to the characteristic insertion site formed by the integration of the IS711 insertion sequence into the alkB gene locus in the Brucella genome. This site is defined by the link region formed by the alkB gene sequence and the IS711 insertion sequence, and can serve as a specific nucleic acid target for Brucella bovis typing detection.

[0028] In this invention, the term "BMEI1162-IS711" refers to the characteristic insertion site formed by the integration of the IS711 insertion sequence in the Brucella genome into the BMEI1162 gene locus. This site is defined by the link region formed by the BMEI1162 gene sequence and the IS711 insertion sequence, and can serve as a specific nucleic acid target for Brucella ovis typing detection.

[0029] In this invention, the term "BS1330_II0657" refers to a specific gene locus (locus tag) derived from the Brucella genome, preferably the BS1330_II0657 gene region located on chromosome 2, and its corresponding nucleic acid sequence or its specific fragment can be used as a specific nucleic acid target for Brucella suis typing detection.

[0030] In this invention, the term "primer" refers to an oligonucleotide sequence that can specifically bind to a target nucleic acid sequence and guide the extension of nucleic acid under the action of DNA polymerase. It typically includes a pair of forward and reverse primers to achieve specific amplification of the target fragment.

[0031] In this invention, the term "probe" refers to an oligonucleotide molecule that can specifically bind to the amplification product and generate a fluorescent signal during nucleic acid amplification. One end of the probe is labeled with a fluorescent reporter group, and the other end is labeled with a quencher group. When the probe is not cleaved or undergoes a conformational change, its fluorescence is suppressed.

[0032] In this invention, the term "self-quenched probe" refers to a class of single-stranded oligonucleotide molecules with a fluorescent group and a quenching group attached to their 5′ and 3′ ends, respectively. When the probe is in a free or unbound state, the spatial distance between the fluorescent group and the quenching group is close or they are energy-coupled, causing the fluorescence energy to be partially or completely quenched. When the probe binds to a complementary target nucleic acid, is cleaved by a nuclease, or undergoes a conformational change, the energy transfer between the fluorescent group and the quenching group is interrupted or the distance increases, thereby releasing a detectable fluorescent signal. Thus, the probe itself can generate and eliminate the signal without the need for an external colorimetric or second reporter system.

[0033] In this invention, the term "TaqMan probe" refers to an oligonucleotide probe with a fluorescent reporter group labeled at the 5' end and a quencher group labeled at the 3' end in a linear structure. During PCR amplification, the probe is cleaved by the 5'→3' exonuclease activity of DNA polymerase, thereby releasing a fluorescent signal.

[0034] In this invention, the term "exogenous internal reference" refers to a nucleic acid molecule that is artificially added during the nucleic acid extraction or amplification process of a sample and has no homology with the target nucleic acid sequence to be tested. It is used to monitor the nucleic acid extraction efficiency, whether the amplification reaction is normal, and whether there are any inhibitory factors.

[0035] In this invention, the term "multiplex fluorescent PCR" refers to a nucleic acid amplification technique that uses multiple sets of primers and probes simultaneously in the same reaction system and performs parallel amplification and detection of multiple target sequences through different fluorescence channels.

[0036] In this invention, the term "kit" refers to any article (e.g., packaging or container) that includes at least one device and comprises the detection reagents as described in this disclosure. The kit may further include instructions for use, supplementary reagents, and / or components or parts used in the methods or steps described in this disclosure.

[0037] In this invention, the term "stabilizer" refers to auxiliary components used to maintain the stability of enzyme activity and overall performance in a PCR reaction system, including but not limited to protein stabilizers, polyols, and surfactants.

[0038] This invention relates to a primer-probe combo product comprising a) to d): a) The primer pairs shown in SEQ ID NO: 1-2 and the probe shown in SEQ ID NO: 3; b) The primer pairs shown in SEQ ID NO: 4-5 and the probe shown in SEQ ID NO: 6; c) The primer pairs shown in SEQ ID NO: 7-8 and the probe shown in SEQ ID NO: 9; d) The primer pairs shown in SEQ ID NO: 10-11 and the probe shown in SEQ ID NO: 12.

[0039] The primer-probe combination products described above can be used to confirm Brucella species and identify Brucella bovis, Brucella ovis, and Brucella swine in a single detection process. This helps to reduce detection steps, improve detection efficiency, and enhance the stability and reliability of detection results. At the same time, it reduces the risk of misjudgment caused by single target mutations, thereby improving the applicability of the detection system in clinical testing and animal quarantine scenarios.

[0040] In some embodiments, it further includes primers and probes for an exogenous internal control. The exogenous internal control is preferably an exogenous nucleic acid molecule added before or during sample nucleic acid extraction. It is not homologous to Brucella spp. and related detection target sequences. Through amplification in conjunction with the corresponding primers and probes, it can be used to monitor whether the nucleic acid extraction process is successful and whether there are inhibitory factors in the amplification system. This helps avoid false negative results caused by extraction failure or amplification abnormalities, thereby improving the reliability and interpretability of the detection results.

[0041] In some preferred embodiments, the primers and probes of the exogenous internal control may include an internal control detection system, such as the primer pair and probe shown in e): e) the primer pair shown in SEQ ID NO: 13-14 and the probe shown in SEQ ID NO: 15. By setting a single internal control system, the flexibility and stability of quality control can be improved while ensuring detection sensitivity; for example, in a multiplex fluorescent PCR system, the internal control probe can be labeled in an independent fluorescent channel to avoid interference with the target signal, thereby facilitating effective monitoring of the entire detection process and further improving the accuracy of the detection system.

[0042] Additionally, it should be noted that, in one respect, the concept of useful primers and probes should include nucleotide sequences having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one or more of the primers or probes shown in SEQ ID NO: 1-15. Modifications of such primers and probes and their ability to be prepared according to standard techniques are also considered.

[0043] The term "%identity" in the context of two or more nucleotide or amino acid sequences refers to two or more sequences or subsequences that are identical or have a specific percentage of the same amino acid residues or nucleotides when compared and aligned for maximum correspondence, as measured by one of the following sequence comparison algorithms or by visual inspection. For example, %identity is relative to the entire length of the coding region of the sequences to be compared.

[0044] For sequence comparisons, a sequence is typically used as a reference sequence, and the test sequence is compared to this sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and subsequence coordinates are specified if necessary, along with the sequence algorithm program parameters. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. Percentage identity can be determined using search algorithms such as BLAST and PSI-BLAST (Altschul et al., 1990, J Mol Biol 215:3, 403-410; Altschul et al., 1997, Nucleic Acids Res25:17, 3389-402).

[0045] Primer and probe modifications can be performed using well-known methods. Modified versions of these primer and / or probe sequences may include, by non-limiting examples, adding one or more nucleotides to the 5' end, adding one or more nucleotides to the 3' end, adding one or more nucleotides to both the 5' and 3' ends, adding a tail, shortening the sequence, lengthening the sequence, shifting the sequence upstream or downstream by several bases, or any combination thereof.

[0046] Base modifications, such as 3'P, 5'P, 5-nitroindole, 2-aminopurine, 8-amino-2'-deoxyadenosine, C-5-propynyl-deoxycytidine, C-5-propynyl-deoxyuridine, 2-amino-2'-deoxyadenosine-5'-triphosphate, 2,6-diaminopurine (2-amino-dA), reversed-dT, reversed-dideoxy-T, hydroxymethyl-dC, iso-dC, 5-methyl-dC, aminoethyl-phenoxazine-deoxycytidine, and locked nucleic acids (LNAs), including at least one mismatched base at one of the bases, or replacing at least one of the bases with an RNA base, can achieve, for example, increased nucleic acid interaction at the 3' end of mutant-specific primers to increase Tm. The addition of stable double-stranded base modifications has a positive effect on PCR, enabling it to be performed at higher temperatures, within which Taq polymerase is known to exhibit maximum activity. Modified probes should retain the ability to distinguish between the mutant and wild-type sites to be detected.

[0047] In some embodiments, the probes are all self-quenching probes. Self-quenching probes known in the art can include various conformations or mechanisms of action; as long as they possess the characteristic of achieving fluorescence quenching and de-quenching through intramolecular or intermolecular energy transfer, they can be considered as "self-quenching probes" of this invention. These probes can be used in real-time fluorescence PCR, melting curve analysis, isothermal amplification, or multiplex amplification systems to achieve qualitative or quantitative detection of nucleic acid targets. The specific form of the self-quenching probe is not limited to any particular structure, but preferably includes, but is not limited to, the following subtypes: stem-loop molecular beacons (MB), linear cleavage probes (TaqMan probes), built-in probes (Scorpion, LUX, or Amplifluor, etc.), and FRET-based dual-probe systems. TaqMan probes are the most preferred.

[0048] In some embodiments, the fluorescent emitting groups of each probe are independently selected from any one of AMCA, Pacific Blue, Atto 425, BODIPY FL, FAM, Alexa Fluor 488, TET, JOE, Yakima Yellow, VIC, HEX, Quasar 570, Cy3, NED, TAMRA, ROX, Aqua Phluor 593, Texas Red, Atto 590, Cy5, Quasar 670, and Cy5.5.

[0049] In some embodiments, the quenching groups of each probe are independently selected from any one of BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, and MGB.

[0050] In some embodiments, components a) and b) are packaged together, and components c) and d) are packaged together, each packaged in a separate reaction vessel to form a two-tube detection system. In this configuration, the first reaction system is preferably used for Brucella spp. detection and Brucella bovis typing, while the second reaction system is preferably used for Brucella ovis and Brucella swine typing. This ensures multiplex detection capability while reducing interference between primers and probes in a single-tube system. By allocating different targets to two reaction systems, amplification efficiency and detection sensitivity can be improved to some extent, and the configuration of fluorescence channels and signal interpretation can be optimized, thereby enhancing the overall stability and reproducibility of the detection system.

[0051] The present invention also relates to a kit containing the primer-probe combination product as described above.

[0052] In some embodiments, the kit further contains one or more of the following: nucleic acid extraction reagent, PCR amplification reagent, positive control, negative control, and internal reference template.

[0053] The nucleic acid extraction reagent and PCR amplification reagent can be flexibly configured according to the detection purpose and device structure.

[0054] Furthermore, the nucleic acid extraction reagent may include any one or more of lysis buffer, washing buffer, and elution buffer. The lysis buffer may contain surfactants (such as Triton X-100, Tween-20, SDS), protein denaturants (such as guanidine salts, urea), chelating agents (such as EDTA), and buffer components; the washing buffer may contain appropriate amounts of alcohols or salts to remove impurities; the elution buffer is typically a low-salt buffer or nuclease-free water. The extraction method is not limited to any specific technical route and may include silica-based membrane adsorption, magnetic bead methods, or direct lysis amplification methods, etc. Those skilled in the art can select a suitable extraction scheme based on the detection equipment.

[0055] The positive control can be a standard containing the corresponding target sequence, used to verify the effectiveness of the detection system; the negative control is preferably a template-free system, used to monitor contamination; the internal reference template can be used in conjunction with exogenous internal reference primers and probes to achieve quality control of the nucleic acid extraction and amplification process.

[0056] The PCR amplification reagents may include DNA polymerase, buffer, dNTPs, and Mg. 2+ One or more of the following: stabilizers.

[0057] The DNA polymerase can be a thermostable DNA polymerase, preferably with 5'→3' exonuclease activity to be suitable for the TaqMan probe detection system, such as Taq DNA polymerase, hot-start Taq DNA polymerase, HotStart Taq, Tth DNA polymerase or modified enzymes thereof; in some embodiments, engineered polymerases with higher amplification efficiency or inhibition resistance can also be selected.

[0058] The buffer system is preferably used to maintain a suitable ionic strength and pH environment, and may include Tris-HCl buffer, KCl, (NH4)2SO4, or a combination thereof. The pH of the buffer system is preferably 7.5–9.0, more preferably about 8.0–8.8, or any intermediate value thereof, to facilitate the maintenance of DNA polymerase activity and the stability of the amplification reaction. The Mg... 2+ It can exist in MgCl2 or its equivalent form, and its concentration can be adjusted according to the primer and probe system, for example, from 1 mM to 6 mM or any intermediate value, to optimize amplification efficiency and specificity.

[0059] The stabilizer can be a component used to improve enzyme stability and the system's resistance to interference, such as protein stabilizers (e.g., bovine serum albumin), polyols (e.g., glycerol, trehalose, sorbitol), nonionic surfactants (e.g., Tween-20, NP-40), and other additives that improve amplification performance (e.g., betaine, DMSO). By introducing these stabilizers, the stability of the reagents during storage, transportation, and repeated freeze-thaw cycles can be improved to a certain extent, and the inhibitory effect of complex sample matrices on the PCR reaction can be reduced, thereby improving the repeatability and reliability of the detection results.

[0060] The components in the kit can be packaged in the form of solutions, solids, or test strips. Premixed or lyophilized forms are preferred to allow for rapid reconstitution and stable storage. In some preferred embodiments, at least one component of the reagent or kit is a solid, including at least one of lyophilized microspheres, lyophilized cakes, lyophilized powders, or spots formed on the surface of a solid carrier. Therefore, components required for nucleic acid amplification (and preferably nucleic acid detection) can be provided in lyophilized form, particularly various enzymes, nucleic acid components, and reaction buffer components. In this way, the nucleic acid amplification (and preferably nucleic acid detection) process can be started directly in a user-friendly manner by adding the sample to be quantified and optionally other required components.

[0061] The present invention also relates to the use of the primer-probe combination product described above in the preparation of a kit for differentiating infections of Brucella bovis, Brucella ovis, and Brucella swine.

[0062] The primer-probe combination, through synergistic design targeting both conserved targets and species-specific targets of Brucella, generates corresponding fluorescent signals during nucleic acid amplification, thereby enabling the identification of Brucella species and the typing of different species in a single detection. The kit developed based on this combination is suitable for detecting various types of samples, including blood, tissue, milk, and abortion products, and can be used in clinical diagnosis, animal disease monitoring, and quarantine screening applications.

[0063] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these embodiments are only used to illustrate the technical content of the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the specific experimental conditions in the following embodiments are given priority reference to the guidelines provided in this specification, or may be carried out according to generally accepted experimental manuals or conventional experimental conditions, or other experimental methods known in the art, or according to the conditions recommended by the relevant reagent or instrument manufacturers. In specific embodiments, unless otherwise specified, minor deviations within the weighing accuracy range are allowed for the measurement parameters involving raw material components; reasonable deviations due to instrument detection accuracy or operational accuracy are also allowed for parameters such as temperature and time.

[0064] Example 1: Primer-Probe Set Design and Channel Assignment This embodiment provides a channel allocation scheme for multiplex fluorescent PCR.

[0065] Example channel settings are as follows:

[0066] Primer and probe sequences, working concentrations, and amplification fragment lengths can be set as follows:

[0067] Example 2: Kit Composition This embodiment provides a detection kit composition, comprising: • 2×PCR mixture (containing hot-start DNA polymerase, buffer, dNTPs, Mg) 2+ (stabilizers, etc.) • Primer and probe mixture (containing primers and probes from each group in Example 1); • Positive control (can be a plasmid mixture containing each target fragment or a standard); • Negative control (without template); • Internal reference template Example 3: Detection Process 1. Nucleic acid extraction: Extraction using magnetic bead method / column method / boiling method; 2. Reaction system (25 μL): 12.5 μL of 2×Mix, 7.5 μL of primer-probe Mix, 5 μL of template, and water to 25 μL; 3. Amplification procedure: Fluorescence signals were collected at 94℃ for 10 seconds, 60℃ for 30 seconds, and 72℃ for 15 seconds for 40 cycles, with the fluorescence signal collected at 60℃. 4. Interpretation threshold: Set baseline and threshold. Ct≤40 is considered positive; Ct between 38-40 is considered suspicious and requires retesting; Ct>40 is considered negative.

[0068] Example 4: Interpretation Rules One interpretation logic of this invention is as follows: (A) belongs to the determination • If the horizontal target channel (IS711) shows typical S-shaped amplification and Ct≤40, it is judged as Brucella spp. positive; (B) Subtype determination (based on a positive result) • Bovine fabric: positive for bovine fabric typing targets, negative for sheep fabric and pig fabric typing targets; •Wool fabric: Positive for genotyping targets in wool fabric, negative for genotyping targets in bovine and swine fabric; • Swine bromide: Positive for swine bromide typing targets, negative for bovine bromide and sheep bromide typing targets; • If two or more subtype targets are positive at the same time, it is considered an abnormal result, and it is recommended to retest and use orthogonal methods for confirmation.

[0069] Depend on Figure 2 It can be seen that different targets can form typical S-shaped amplification curves in their corresponding channels, and there are no obvious abnormal amplification signals in non-corresponding channels. This indicates that the two-tube detection system established in this invention can achieve Brucella identification and typing of Brucella bovis, Brucella ovis, and Brucella swine.

[0070] Example 5: Sensitivity Validation of the Multiplex Fluorescent PCR Detection System To verify the sensitivity of the multiplex fluorescent PCR detection system established in this invention, evaluate the minimum detection capability of each target under multiplex amplification conditions, and investigate whether there is significant amplification competition or mutual interference between different primers and probes in the multiplex reaction system, the sensitivity of the kit of this invention is verified.

[0071] I. Experimental Materials 1. Recombinant plasmids containing the IS711 target fragment were selected as IS711 positive standards; recombinant plasmids containing the alkB-IS711 linkage region (cow's cloth) target fragment were selected as alkB-IS711 linkage region (cow's cloth) positive standards; recombinant plasmids containing the BMEI1162-IS711 linkage region (sheep's cloth) target fragment were selected as BMEI1162-IS711 linkage region (sheep's cloth) positive standards; and recombinant plasmids containing the BS1330_II0657 (swine's cloth) target fragment were selected as BS1330_II0657 (swine's cloth) positive standards. The targets correspond to different detection channels in the multiplex fluorescent PCR system of this invention.

[0072] 2. After the recombinant plasmid is sequenced to verify that the inserted sequence is correct, the concentration is determined by nucleic acid quantification method and converted into copy number concentration according to molecular weight.

[0073] 3. Each positive standard was serially diluted with sterile water to prepare nucleic acid templates at different concentration levels.

[0074] 4. The concentration gradients used in this embodiment include: 1000 copies / reaction, 500 copies / reaction, 100 copies / reaction, 50 copies / reaction, 20 copies / reaction, and 10 copies / reaction.

[0075] 5. A negative control was set up, using sterile water that does not contain the target nucleic acid as a template.

[0076] 6. The internal control template is added to each reaction system in a fixed amount to monitor the amplification process and the effectiveness of the reaction system.

[0077] II. Experimental Methods 1. Sensitivity testing of single-system systems Positive standards for target IS711, alkB-IS711 linker region (cow's cloth), BMEI1162-IS711 linker region (sheep's cloth), and BS1330_II0657 (swine cloth) were taken and added to the corresponding singlet fluorescent PCR reaction systems according to the above concentration gradient for amplification and detection. Twenty replicates were set up for each concentration level, and a negative control was also included.

[0078] By statistically analyzing the number of positive detections at different concentrations, the limit of detection for each target under single amplification conditions was evaluated.

[0079] 2. Multiple system sensitivity testing The IS711 positive standard, alkB-IS711 linker region (cow's cloth) positive standard, BMEI1162-IS711 linker region (sheep's cloth) positive standard, and BS1330_II0657 (swine's cloth) were added to two identical multiplex fluorescent PCR reaction systems at their respective concentrations. The IS711 positive standard and alkB-IS711 linker region (cow's cloth) positive standard were in the same reaction system, as were the BMEI1162-IS711 linker region (sheep's cloth) positive standard and BS1330_II0657 (swine's cloth) positive standards. A fixed amount of internal control template was also added for multiplex amplification detection. Twenty replicates were set up for each concentration level, and a negative control was included.

[0080] The sensitivity of the multiplex fluorescent PCR system of this invention under the condition of simultaneous amplification of multiple targets was evaluated by statistically analyzing the number of positive detections of each target at different concentration levels in the multiplex system.

[0081] III. Result Judgment Criteria When a typical S-shaped amplification curve appears in a detection channel and the cycle threshold (Ct value) falls within the preset judgment range, the target corresponding to that channel is judged to be positive; if no obvious amplification curve appears or the Ct value exceeds the preset judgment range, it is judged to be negative. For the negative control, all detection channels should show no amplification signal, and the internal control channel should show normal amplification.

[0082] In this embodiment, the lowest template concentration corresponding to a positive detection rate of not less than 95% in 20 repeated tests at a certain concentration level is taken as the lowest detection limit of the target under the corresponding system conditions.

[0083] IV. Experimental Results 1. Sensitivity results of singlet systems The test results showed that under singlet amplification conditions: (1) At concentration levels of 1000 copies / reaction, 500 copies / reaction and 100 copies / reaction, all 20 repeated tests of the target IS711 were positive, with a detection rate of 100%; at a concentration level of 50 copies / reaction, 19 out of 20 repeated tests were positive, with a detection rate of 95%; at a concentration level of 20 copies / reaction, 16 out of 20 repeated tests were positive, with a detection rate of 80%; at a concentration level of 10 copies / reaction, the positive detection rate further decreased.

[0084] (2) The target alkB-IS711 linker region (nucleo) was positive in 20 repeated tests at concentration levels of 1000 copies / reaction, 500 copies / reaction and 100 copies / reaction, with a detection rate of 100%; at a concentration level of 50 copies / reaction, 18 out of 20 repeated tests were positive, with a detection rate of 90%; at a concentration level of 20 copies / reaction, the positive detection rate further decreased.

[0085] (3) The BMEI1162-IS711 linker region (wool cloth) of the target was positive in 20 repeated tests at concentration levels of 1000 copies / reaction, 500 copies / reaction and 100 copies / reaction, with a detection rate of 100%; at the concentration level of 50 copies / reaction, 19 out of 20 repeated tests were positive, with a detection rate of 95%; at the concentration level of 20 copies / reaction, 15 out of 20 repeated tests were positive, with a detection rate of 75%; at the concentration level of 10 copies / reaction, the positive detection rate further decreased.

[0086] (4) Target BS1330_II0657 (swine cloth) was positive in 20 repeated tests at concentration levels of 1000 copies / reaction, 500 copies / reaction and 100 copies / reaction, with a detection rate of 100%; at a concentration level of 50 copies / reaction, 18 out of 20 repeated tests were positive, with a detection rate of 90%; at a concentration level of 20 copies / reaction, the positive detection rate further decreased.

[0087] 2. Sensitivity results of multiple systems The test results further show that, under the multiplex fluorescent PCR amplification conditions of this invention: (1) At concentration levels of 1000 copies / reaction and 500 copies / reaction, all 20 repeated tests of the target IS711 were positive; at concentration level of 100 copies / reaction, all 20 repeated tests were positive, with a detection rate of 100%; at concentration level of 50 copies / reaction, 18 out of 20 repeated tests were positive, with a detection rate of 90%; at concentration level of 20 copies / reaction, the detection rate further decreased.

[0088] (2) The target alkB-IS711 linker region (nucleo) was positive in 20 repeated tests at concentration levels of 1000 copies / reaction, 500 copies / reaction, 100 copies / reaction and 50 copies / reaction, with a detection rate of 100%; at a concentration level of 20 copies / reaction, 18 out of 20 repeated tests were positive, with a detection rate of 90%; at a concentration level of 10 copies / reaction, the positive detection rate further decreased.

[0089] (3) At concentration levels of 1000 copies / reaction, 500 copies / reaction and 100 copies / reaction, 19 out of 20 replicates of the target BMEI1162-IS711 linker (wool cloth) were positive, with a detection rate of 95%; at a concentration level of 50 copies / reaction, 17 out of 20 replicates were positive, with a detection rate of 85%; at a concentration level of 20 copies / reaction, the positive detection rate further decreased.

[0090] (4) Target BS1330_II0657 (swine swine) was positive in 20 replicate tests at concentration levels of 1000 copies / reaction, 500 copies / reaction and 100 copies / reaction, with a detection rate of 100%; at a concentration level of 50 copies / reaction, 19 out of 20 replicate tests were positive, with a detection rate of 95%; at a concentration level of 20 copies / reaction, the positive detection rate further decreased.

[0091] Based on the above results, the limits of detection for the targets are inconsistent under multiplex amplification conditions. Under the conditions of this embodiment, the limit of detection for target IS711 under multiplex amplification conditions is 100 copies / reaction; the limit of detection for target alkB-IS711 linker region (bovine cloth) under multiplex amplification conditions is 50 copies / reaction; the limit of detection for target BMEI1162-IS711 linker region (sheep cloth) under multiplex amplification conditions is 100 copies / reaction; and the limit of detection for target S1330_II0657 (swine cloth) under multiplex amplification conditions is 50 copies / reaction.

[0092] Depend on Figure 3 As can be seen, as the template concentration decreases, the amplification curves of each target shift backward as a whole, and the Ct value increases accordingly. However, when the corresponding minimum detection limit is reached, a typical amplification curve that can still be interpreted can still be formed, indicating that the multiplex fluorescent PCR system established in this invention has good sensitivity.

[0093] Example 6: Specificity Verification I. To verify the specificity of the alpha-binding target (alkB-IS711 linker region), the following verification was conducted in this embodiment: 1. Target strain coverage verification: Select 20 or more Brucella bovis strains with clear sources (covering different regions / biotypes or representative strains) for testing. All strains should be positive for Brucella bovis target. 2. Exclusionary verification by close relatives: Select *Brucella melitensis* n≥20, *Brucella suis* n≥20, and other *Brucella* species / biotypes (such as *Brucella canis*, *Brucella ovis*, etc., as needed) for testing; bovine *Brucella* target should be negative; 3. External cross-reactivity verification: Select bacteria commonly mixed with clinical / livestock samples (such as Escherichia coli, Yersinia pestis, Salmonella, Vibrio cholerae, etc.) and potentially interfering species n≥30 for testing, and the result should be negative; 4. Mixed template verification: Mix sheep fabric DNA with bovine / swine fabric DNA at different copy ratios (e.g., 1:1, 1:10, 10:1) to verify that the DNA can still be correctly detected and genotyped under multiplex systems. 5. Threshold boundary verification: For low-copy diluted samples, verify repeatability when Ct is close to the threshold range, and establish a "suspicious retest" interpretation rule.

[0094] The results showed that, under the above verification conditions, the bovine Brucella typing target was positive for bovine Brucella strains and negative for non-bovine Brucella and extragenus bacteria, indicating that the typing target has good specificity and practicality.

[0095] II. To verify the specificity of the wool morphology target (BMEI1162-IS711 linker region), the following verification was conducted in this embodiment: 1. Target strain coverage verification: Select 20 or more Brucella oviduct strains with clear sources (covering different regions / biotypes or representative strains) for testing. All strains should be positive for Brucella oviduct target. 2. Exclusionary verification by close relatives: Select *Brucella bovis* n≥20, *Brucella ovis* n≥20, and other *Brucella* species / biotypes (such as *B. canis*, *B. ovis*, etc., as needed) for testing; the *Brucella swine* target should be negative; 3. External cross-reactivity verification: Select bacteria commonly mixed with clinical / livestock samples (such as Escherichia coli, Yersinia pestis, Salmonella, Vibrio cholerae, etc.) and potentially interfering species n≥30 for testing, and the result should be negative; 4. Mixed template verification: Swine fabric DNA was mixed with bovine fabric and sheep fabric DNA at different copy ratios (e.g., 1:1, 1:10, 10:1) to verify that the DNA could still be correctly detected and genotyped under multiplex systems. 5. Threshold boundary verification: For low-copy diluted samples, verify repeatability when Ct is close to the threshold range, and establish a "suspicious retest" interpretation rule.

[0096] The results showed that, under the above verification conditions, the Brucella genotyping target was positive for Brucella spp. strains and negative for non-Brucella spp. and other bacteria, indicating that the genotyping target has good specificity and practicality.

[0097] III. To verify the specificity of the porcine genotyping target (BS1330_II0657), the following verification was conducted in this embodiment: 1. Target strain coverage verification: Select 20 or more Brucella suis strains with clear sources (covering different regions / biotypes or representative strains) for testing. All strains should be positive for Brucella suis target. 2. Exclusionary verification by close relatives: Select *Brucella bovis* n≥20, *Brucella suis* n≥20, and other *Brucella* species / biotypes (such as *B. canis*, *B. ovis*, etc., as needed) for testing. The target of *Brucella suis* should be negative. 3. External cross-reactivity verification: Select bacteria commonly mixed with clinical / livestock samples (such as Escherichia coli, Yersinia pestis, Salmonella, Vibrio cholerae, etc.) and potentially interfering species n≥30 for testing, and the result should be negative; 4. Mixed template verification: Mix bovine fabric DNA with bovine and porcine fabric DNA at different copy ratios (e.g., 1:1, 1:10, 10:1) to verify that the DNA can still be correctly detected and genotyped under multiplex systems. 5. Threshold boundary verification: For low-copy diluted samples, verify repeatability when Ct is close to the threshold range, and establish a "suspicious retest" interpretation rule.

[0098] Depend on Figure 4 As can be seen, the target bacteria exhibited typical amplification curves in the corresponding samples, while no obvious amplification signals were observed in non-target strains and exogenous bacteria. This indicates that the multiplex fluorescent PCR detection system established in this invention has good specificity for Brucella typing targets and a low risk of cross-reaction. Under the above verification conditions, the swine Brucella typing target only produced a positive signal for swine Brucella, and no amplification was detected for other Brucella strains and exogenous bacteria, further demonstrating that this target has strong discriminative ability in typing detection and has good application value.

[0099] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A primer-probe combo product, characterized in that, Including a)~d): a) The primer pairs shown in SEQ ID NO: 1-2 and the probe shown in SEQ ID NO: 3; b) The primer pairs shown in SEQ ID NO: 4-5 and the probe shown in SEQ ID NO: 6; c) The primer pairs shown in SEQ ID NO: 7-8 and the probe shown in SEQ ID NO: 9; d) The primer pairs shown in SEQ ID NO: 10-11 and the probe shown in SEQ ID NO:

12.

2. The combined product according to claim 1, characterized in that, It also includes primers and probes for exogenous internal references.

3. The combined product according to claim 2, characterized in that, The primers and probes of the exogenous internal reference include the primer pairs shown in SEQ ID NO: 13-14 and the probe shown in SEQ ID NO:

15.

4. The combined product according to any one of claims 1-3, characterized in that, All probes are self-quenching probes, with TaqMan probes being preferred.

5. The combined product according to any one of claims 1-3, characterized in that, Components a) and b) are packaged together, and components c) and d) are packaged together, and then packaged separately in different reaction containers to form a two-tube detection system.

6. A reagent kit, characterized in that, A product containing the primer-probe combination according to any one of claims 1-5.

7. The reagent kit according to claim 6, characterized in that, It also contains one or more of the following: nucleic acid extraction reagent, PCR amplification reagent, positive control, negative control, and internal reference template.

8. The reagent kit according to claim 7, characterized in that, The PCR amplification reagents include DNA polymerase, buffer, dNTPs, and Mg. 2+ One or more of the following: stabilizers.

9. The reagent kit according to any one of claims 6-8, characterized in that, At least one component of the kit is a solid, and the solid includes at least one of lyophilized microspheres, lyophilized cakes, lyophilized powder, or spots formed on the surface of a solid carrier.

10. The use of the primer-probe combination product according to any one of claims 1-5 in the preparation of a kit for differentiating infections of Brucella bovis, Brucella ovis, and Brucella swine.

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