A pair of pcr primers, a kit for granulomatous mastitis etiology classification and purposes thereof

Abstract

This invention belongs to the field of biological detection technology, specifically relating to a PCR primer pair, kit, and its uses for the etiological classification of granulomatous mastitis. This invention provides a real-time PCR detection kit containing four primer pairs, targeting: (1) group: CKC-specific targets; (2) genus: Corynebacterium (3) General: General bacterial targets; (4) Human samples. GM pathogen classification is achieved through hierarchical combination interpretation of qPCR results. The detection method of this invention has high specificity, can distinguish four types of GM through a single detection, has a short detection cycle, high sensitivity and accuracy, and can also achieve objective differentiation between bacterial and non-bacterial infections by setting a Cq cutoff value. It has good application prospects in the detection and treatment of granulomatous mastitis.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a PCR primer pair, kit, and its uses for the etiological classification of granulomatous mastitis. Background Technology

[0002] Granulomatous mastitis (GM) is a chronic inflammatory disease of the breast characterized by non-caseating granulomas. Its global incidence has been rising in recent years, and it is clinically difficult to cure and prone to recurrence. The etiology of GM can be divided into two main categories: (1) Bacterial GM, with Corynebacterium spp. as the main pathogens, including Corynebacterium kuribda complex (…). Corynebacterium kroppenstedtii The highest detection rate was for complex CKC, which includes C. kroppenstedtii , C. parakroppenstedtii , C. pseudokroppenstedtii etc.; in addition, closely related bacteria (such as C. tuberculostearicum , C. glucuronolyticum (2) Non-bacterial GM may be mediated by autoimmune mechanisms. The treatment strategies for the two types of GM are fundamentally different: bacterial GM requires antibiotics based on drug sensitivity results, while antibiotics are ineffective for non-bacterial GM. Therefore, accurate etiological classification of GM is a prerequisite for guiding rational clinical treatment.

[0003] However, existing methods for detecting GM pathogens have significant limitations. Traditional bacterial culture methods have low detection rates for Corynebacterium spp., are time-consuming, and have demanding culture conditions for some species. While conventional PCR and sequencing methods have high sensitivity, they are complex to operate, have long detection cycles, and are costly, making it difficult to meet the needs of rapid clinical typing and diagnosis. For example, sequencing-dependent PCR (16S rRNA PCR + Sanger sequencing) can detect bacterial DNA in culture-negative specimens, but the sequencing process is time-consuming (usually 1-2 days) and cannot provide quantitative results. Metagenomic next-generation sequencing (mNGS) has a high pathogen detection rate (reported to reach 60.0%), but the accuracy of mNGS is affected by database completeness and bioinformatics analysis procedures, and the detection cost is high, making it difficult to use routinely. Targeted... C. kroppenstedtii The single-strain qPCR method is only for C. kroppenstedtii Single type, not covered C. parakroppenstedtii , C. pseudokroppenstedtii CKC members and other pathogens Corynebacterium Due to the limited number of genera and species, a complete pathogenic hierarchical classification of GM is not possible.

[0004] Therefore, there is an urgent need in this field to develop a detection method that is easy to operate, highly sensitive, highly specific, and can quickly distinguish the types of GM bacterial infections, so as to achieve accurate etiological classification of GM and provide technical support for the development of individualized treatment plans in clinical practice. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a PCR primer pair, kit, and its uses for the etiological classification of granulomatous mastitis.

[0006] This invention provides a PCR primer pair for the etiological classification of granulomatous mastitis, comprising: a CKC-specific primer pair and a Corynebacterium genus-specific primer pair; the nucleotide sequence of the CKC-specific primer pair is shown in SEQ ID NO.1-2.

[0007] Preferably, the nucleotide sequences of the Corynebacterium-specific primer pairs are shown in SEQ ID NO. 3-4.

[0008] Preferably, it also includes bacterial universal primer pairs and human housekeeping gene primer pairs.

[0009] Preferably, the nucleotide sequence of the bacterial universal primer is shown in SEQ ID NO.5-6.

[0010] Preferably, the human housekeeping genes include β-actin, GAPDH, and 18S rRNA, and the nucleotide sequence of the β-actin primer pair is shown in SEQ ID NO.7-8.

[0011] This invention provides the use of the PCR primer pairs as described in any of the preceding claims for the etiological classification of granulomatous mastitis in the preparation of a kit for the etiological classification of granulomatous mastitis.

[0012] Preferably, the etiological classification includes: CKC infection and non-CKC rod-shaped bacillus infection.

[0013] Preferably, the basis for the etiological classification includes: performing PCR amplification reactions on the test samples using PCR primers; When the CKC-specific primer pair produces a specific amplification product, it is determined to be CKC positive; when the CKC-specific primer pair produces no specific amplification product, but the Corynebacterium genus-specific primer pair produces a specific amplification product, it is determined to be non-CKC Corynebacterium positive. The PCR amplification reaction conditions included: denaturation at 95℃ for 10 seconds, annealing at 55-65℃ for 30 seconds, extension at 72℃ for 30 seconds, and 40 cycles.

[0014] Preferably, the etiological classification also includes: bacterial infections other than Corynebacterium and non-bacterial infections; The criteria for pathogen classification also include: when the quantitative cycle number Cq of the PCR amplification reaction using the universal bacterial primer pair is ≤22.43, and when the CKC-specific primer pair and the Corynebacterium genus-specific primer pair produce no specific amplification products, the result is determined to be positive for bacteria other than Corynebacterium. When the quantitative cycle number Cq of the PCR amplification reaction using the universal bacterial primer pair is greater than 22.43, and the reaction of the CKC specific primer pair and the Corynebacterium genus specific primer pair produces no specific amplification product, the result is determined to be bacterial negative. And / or, the kit may also include amplification reagents, positive controls, and negative controls.

[0015] Preferably, the PCR is qPCR.

[0016] The present invention provides a kit comprising PCR primer pairs for etiological classification of granulomatous mastitis as described in any of the preceding claims.

[0017] This invention provides a multi-tube single-tube quantitative real-time PCR (qPCR) detection kit and its detection method for pathogen classification of GM pus specimens by screening primer sequences. The kit contains 4 pairs of primers, targeting: (1) group: CKC specific target ( rpoB (2) Genus: Corynebacterium Specific targets ( 16S rRNA (3) Universal: Bacterial universal targets (genes); (4) Universal targets: Bacterial universal targets (genes); (5) Universal targets: Bacterial universal targets (genes); (6) Universal targets: Bacterial universal targets (genes); (7) Universal targets: Bacterial universal targets (genes); (8) Universal targets: Bacterial universal targets (genes); (9) Universal targets: Bacterial universal targets (genes); (10) Universal targets: Bacterial universal targets (genes 16S rRNA (4) Human samples (gene common segments); β-actin (Genes). GM pathogen classification is achieved through hierarchical combination interpretation of qPCR results.

[0018] The kit and detection method of the present invention have the following beneficial effects: (1) High specificity, which can simultaneously cover the entire spectrum of CKC, solving the problem that existing qPCR technology only targets C. kroppenstedtii (1) It has the ability to classify pathogens hierarchically and distinguish four types of GM in a single test; (2) It has a high detection rate and accuracy and a low risk of missed detection; (3) It has a short turnaround time (≤4 hours) and high sensitivity; (4) It has a quality control system (β-actin) that includes sample validity; (5) It has established a universal primer Cq cutoff value (22.43) to achieve objective distinction between bacterial infection and non-bacterial infection.

[0019] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0020] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0021] Figure 1 ROC curve plotted for clinical pus sample testing; Figure 2 This is an electrophoresis image of PCR for specific detection using 12 representative bacterial strains as test samples; lane M represents the DNA marker, and lane 1 represents Corynebacterium kuribda. C. kroppenstedtii (CKC), lane 2 indicates Corynebacterium paraknegiformis. C. parakroppenstedtii (CKC), lane 3 indicates *Corynebacterium mucinum*. C. mucifaciens (Non-CKC Corynebacterium), lane 4 indicates Corynebacterium glutamicum. C. glutamicum (Non-CKC Corynebacterium), lane 5 indicates Beijing Corynebacterium. C. pekinense (Non-CKC Corynebacterium), lane 6 indicates non-mycolic acid Corynebacterium. C. amycolatum (Non-CKC rod-shaped bacteria), lane 7 indicates abscess mycobacteria. M. abscessus (Closely related genus), lane 8 indicates BCG vaccine. M. bovis BCG (closely related genus), lane 9 indicates Micrococcus luteus. M. luteus (Distant bacteria), lane 10 represents Pseudomonas aeruginosa. P. aeruginosa (Distant bacteria), lane 11 represents Staphylococcus aureus. S. aureus (Distant bacteria), lane 12 indicates Staphylococcus epidermidis S. epidermidis (Distant bacteria), lane N indicates negative control NTC; Figure 2 A is an electrophoresis diagram of CKC-specific primer amplification. Figure 2 B is an electrophoresis diagram of amplification using Corynebacterium-specific primers. Figure 2 C is the electrophoresis diagram of bacterial universal primer amplification. Figure 2 D is the electrophoresis diagram of β-actin primer amplification. Detailed Implementation

[0022] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.

[0023] Sample pretreatment methods: Pus sample collection: During surgical drainage, use a sterile syringe to draw pus and immediately transfer it to a sterile 1.5 mL centrifuge tube to obtain the original pus solution. Store at 4°C and process within 24 hours.

[0024] Thermal lysis method (sample: pus fluid): Transfer 100 μL of pus fluid to a sterile 1.5 mL centrifuge tube, centrifuge at 12,000 × g for 2 minutes, aspirate and discard the supernatant, add an equal volume of sterile enzyme-free water, and vortex for 30 seconds to mix. Transfer to a PCR tube and thermally lyse at 100°C for 10 minutes. Cool on ice at 4°C for 2 minutes, then centrifuge briefly for 5 seconds. Use the supernatant as a qPCR template for immediate use or short-term storage at -20°C.

[0025] Thermal lysis method (sample is bacterial suspension): Pick a single colony and culture it in liquid culture medium. When the bacteria grow to the logarithmic growth phase, take 100 μL of bacterial suspension into a PCR tube, thermally lyse it at 100℃ for 10 minutes, and then centrifuge for 5 seconds. The ruptured bacterial cells are at the bottom of the tube. Take the supernatant as a qPCR template.

[0026] Preparation method of DNA extraction solution: Column extraction was performed using the Tiangen Bacterial Genomic DNA Extraction Kit.

[0027] Example 1: A multi-tube single-tube quantitative real-time PCR (qPCR) detection kit and its detection method for pathogen classification of GM pus specimens. I. Reagent Kit This embodiment consists of the following components: a combination of CKC-specific primers ( rpoB -703F / rpoB -989R), Corynebacterium A combination of genus-specific primers (16S-431F / 16S-563R), a combination of universal bacterial primers (357F / 518R), a combination of β-actin primers (β-actin-F / β-actin-R), 2×SYBR GreenqPCR premix containing hot-start DNA polymerase (catalog number Q711-02, Novizan Biotech, Nanjing, China), and a positive control ( C. kroppenstedtii Bacterial DNA, C. amycolatum Bacterial DNA), negative control (nuclease-free water, NTC). See Table 1 for details.

[0028] Table 1. Components of the kit The sequences of the primers in the kit are shown in Table 2.

[0029] Table 2 Primer Sequences II. Detection Methods Based on the type of primer combination, four reaction systems (single tube, total volume 20 μL) were prepared as shown in Table 3, namely CKC detection tube, genus-level detection tube, panbacterial detection tube, and sample validity tube; the combination of forward and reverse primers in the CKC detection tube was the CKC-specific primer combination (SEQ ID NO. 1-2), and the combination of forward and reverse primers in the genus-level detection tube was... Corynebacterium The combination of genus-specific primers (SEQ ID NO.3-4), the combination of forward / reverse primers in the panbacterial detection tube is a combination of universal bacterial primers (SEQ ID NO.5-6), and the combination of forward / reverse primers in the sample validity tube is a combination of β-actin primers (SEQ ID NO.7-8).

[0030] Table 3 Then, using the Archimed R6 real-time PCR instrument, the four tubes were reacted under the same PCR cycle program shown in Table 4.

[0031] Table 4 Each test must include both a positive control and a negative control. Three technical replicates should be performed for each clinical sample.

[0032] III. Judgment of Test Results 1. Determination of sample validity Valid sample: If the difference between the Cq value and NTC of the sample validity tube (ΔCq = Cq_NTC - Cq_sample) is ≥5, the sample is valid and the target results can continue to be interpreted; Invalid sample: The difference between the Cq value and NTC in the sample validity tube is ΔCq < 5, indicating insufficient nucleic acid content or failure of pretreatment. Resampling or re-preparation is required, and the results should not be reported.

[0033] 2. Determination of hierarchical pathogen classification The results of each qPCR tube were used to determine the hierarchical etiological classification of GM, as shown in Table 5.

[0034] Table 5 Note: In the table, "Group (+)" means there is an amplification signal in the CKC detection tube, "Group (-)" means there is no amplification signal in the CKC detection tube, "Genus (+)" means there is an amplification signal in the genus-level detection tube, "Genus (-)" means there is no amplification signal in the genus-level detection tube, "General (+)" means Cq ≤ 22.43 in the pan-bacteria detection tube, and "General (-)" means Cq > 22.43 in the pan-bacteria detection tube.

[0035] CKC-specific and genus-specific primers show no amplification signal in NTC and can be directly interpreted based on the presence or absence of an amplification signal. Universal primers, due to background amplification in NTC (Cq = 28.2–28.3), must be interpreted using a cutoff value of 22.43. Melting curves can help confirm amplification specificity (CKC-specific primers Tm = 88.22 ± 0.09℃; Corynebacterium genus-specific primers Tm = 85.44 ± 0.13℃; bacterial universal primers Tm = 87.08 ± 0.21℃).

[0036] Example 2: Detection of clinical pus samples I. Experimental Methods The reagent kit and detection method of Example 1 were used to test clinical pus samples, and the results were compared with conventional culture methods and preferred culture methods.

[0037] 1. Sample Source Sample source: Sichuan Provincial Women and Children's Hospital and Chengdu 416 Hospital. From January to December 2025, 86 purulent specimens from GM patients diagnosed by imaging and pathology and with indications for surgical drainage were consecutively included. Approved by the Ethics Committee (Approval Nos.: 20230807-220 and KT-2024-078).

[0038] 2. Routine culture method (current method used in hospital laboratory departments) Routine culture refers to the standard bacterial culture procedure currently used by the laboratories of participating hospitals for pathogen isolation from breast pus samples, mainly including routine culture media such as blood agar plates, MacConkey agar, and chocolate agar. The specific procedure is as follows: The GM pus specimen was spread and inoculated on the above-mentioned plate, and cultured under aerobic conditions according to the department's routine procedures. The incubation temperature is generally 35-37 ℃. Colony growth should be observed daily, with a maximum duration of 3-7 days, to detect slow-growing lipophilic bacteria such as... Corynebacterium kroppenstedtii ; Suspected colonies were subjected to Gram staining and routine biochemical identification, and species-level identification was performed using MALDI-TOF MS when necessary.

[0039] This routine culture procedure conforms to the basic principles of bacterial culture from breast pus samples as outlined in the "Guidelines for Laboratory Work in Clinical Microbiology and Infectious Diseases in China" and CLSIM22. In this invention, the detection rate data from routine culture were primarily derived from a review of medical records from the Sichuan Provincial Maternal and Child Health Hospital.

[0040] 3. Optimal Cultivation Method The "optimized culture method" in this invention was previously published by the applicant in [Journal Name]. European Journal of Clinical Microbiology and Infectious DiseasesThe improved culture method established in the article (link below: https: / / pubmed.ncbi.nlm.nih.gov / 40711641 / ) aims to improve the isolation success rate and shorten the culture time for Corynebacterium kuribda (CKC). Specific conditions are as follows: CAMHB (Cation-Adjusted Mueller-Hinton Broth) is used as the basal medium, supplemented with 3.3% horse serum, β-NAD, and 1% Tween 80 to provide lipids and growth factors, thereby accelerating CKC growth; the culture dishes after plating are sealed with sealing film and incubated at 35-37 ℃. Single colonies can be observed in approximately 36.8 h. Each sample is plated three times and incubated for 7 days.

[0041] This improved culture method has been demonstrated in previous studies to have a higher detection rate and better drug sensitivity evaluation ability for Corynebacterium in GM pus samples than the conventional culture method, and can be used as a reference control for the qPCR method of this invention.

[0042] 4. Evaluation method for the detection results of the qPCR method of the present invention All isolated strains from the pus samples were further identified by 16S rRNA sequencing to confirm their affiliation with the CKC complex, non-CKC corynebacteria, or other bacteria. Given the lack of a universally accepted single gold standard for detecting CKC / corynebacterium infection, this embodiment does not use culture as the sole gold standard, but rather employs a composite reference standard to determine sample status. This embodiment uses modified culture medium results combined with qPCR positive product sequencing as a reference method to systematically compare the qPCR results of this invention, demonstrating the accuracy and reliability of the detection results. The qPCR detection rate, modified culture method detection rate, and false negative rate used below are all statistically obtained based on this reference method, objectively reflecting the performance improvement of qPCR compared to the current best culture technology.

[0043] Detection rate = (Number of positive samples / Total number of samples) × 100%.

[0044] The false negative rate of the culture method = (number of samples that are positive by qPCR and negative by culture method) / (number of samples that are positive by qPCR method) × 100%.

[0045] qPCR false negative = number of false negative samples by qPCR ÷ number of positive composite samples × 100%; Among them, a composite positive result means that the result is positive by the qPCR method of this invention and confirmed by sequencing; or that the result is positive by the optimized culture method and confirmed by sequencing. False negatives in qPCR: The qPCR method of this invention is negative, but after optimized culture and sequencing, it is identified as positive.

[0046] II. Experimental Results 1. Clinical sample detection results based on the qPCR detection method of this invention (1) Sample validity: β-actin detection of all 86 specimens was valid (Cq range 17.03~26.69, mean 20.78±2.32; ΔCq ≥5).

[0047] (2) ROC curve as shown Figure 1 As shown, the analysis results were: AUC=0.887 (standard error SE=0.045, 95% CI: 0.799~0.975, P<0.001), cutoff value Cq=22.43, Youden index=0.641, sensitivity was 74.6%, and specificity was 89.5%. The qPCR detection results of this invention are shown in Table 6.

[0048] Table 6 2. Comparison with conventional culture methods and optimized culture methods The comparison results between the detection method of this invention and the culture method are shown in Table 7. Conventional culture methods generally require 3-7 days to detect lipophilic, slow-growing bacteria such as *Corynebacterium kuribda*, and the false negative rate is relatively high. In contrast, the detection method of this invention ensures that samples negative by qPCR are all negative after culture identification, meaning the false negative rate of the qPCR method is 0, and the false negative rate of the qPCR method of this invention is 0.0%. Furthermore, compared with culture methods and preferred culture methods, the detection rate is higher, and the risk of false negatives is extremely low.

[0049] Table 7 Comparison of qPCR detection and culture method results Note: Detection rate = number of positive samples / total number of samples × 100%; culture method false negative rate = number of samples that are positive by qPCR and negative by culture method / number of samples that are positive by qPCR × 100%.

[0050] Therefore, the detection method of the present invention has a high detection rate, an extremely low false negative rate, and a high detection rate when used to detect clinical pus samples.

[0051] Example 3: Retrospective etiological classification of GM patients Of the 86 patients in Example 2, 69 with complete medical records were retrospectively classified by etiology, and the results are shown in Table 8. There were no statistically significant differences among the groups in age, lesion size, treatment duration, frequency of hospitalization, and incidence of sinus tracts / fistulas (P > 0.05), demonstrating that clinical characteristics cannot distinguish the type of infection, and etiological classification has independent clinical value.

[0052] Table 8 Note: The qPCR method in Table 8 is the detection method of Example 1; n represents the number of samples for the corresponding infection type detected by the qPCR method.

[0053] The comparison results show that the detection rates of different methods vary significantly. The detection rate of the present invention is significantly higher than that of the conventional culture method and the modified culture method. This is mainly because the detection method of the present invention has higher sensitivity.

[0054] The technical solution of the present invention will be further explained through experiments below.

[0055] Experiment Example 1: Primer Screening Experiment The primer sequences of this invention underwent a four-stage gradual elimination process: I. First Stage: Sequence Analysis and Primer Design 16S rRNA of 4 CKC strains, 6 non-CKC Corynebacterium strains, and 10 closely related / distant strains (a total of 20 strains) were retrieved from the NCBI GenBank database. rpoB and fusA Gene sequences. Information on the 20 strains is shown in Table 9.

[0056] Table 9 Phylogenetic trees were constructed using MEGA 11 with the neighbor-joining method. Multiple sequence alignment was performed using DNAMAN V6.0. Based on the clustering characteristics of CKC in each phylogenetic tree, the phylogenetic tree was determined. rpoB The design strategy used genes as CKC-specific targets and 16S rRNA as genus-specific targets. Based on this, 20 pairs of candidate specific primers were designed according to the MIQE guidelines (9 pairs of CKC-specific primers: 6 pairs containing 16S rRNA, ...). rpoB 3 pairs; 11 pairs of Corynebacterium-specific primers: including 5 pairs of 16S rRNA. fusA (6 pairs). Additionally, 10 pairs of universal bacterial primers were selected as candidates from the literature.

[0057] The nucleotide sequences of 20 candidate specific primers are shown in Table 10-11. The nucleotide sequences of universal bacterial primers are shown in Table 12.

[0058] Table 10. Nine candidate CKC-specific primer pairs Table 11 11 pairs of candidate Corynebacterium spp. specific primers Table 12 10 pairs of universal primers for bacteria II. Second Stage: Primer-BLAST Pre-screening The 20 candidate specific primers were evaluated for specificity using NCBI Primer-BLAST. Based on the predicted number of off-target bacteria and the location of mismatches, 14 primers predicted to have non-specific amplification were eliminated, and 6 primers were retained for the experimental validation stage.

[0059] The six pairs of candidate specific primers retained in the second phase are as follows: 3 pairs of CKC-specific candidate primers: rpoB -703F / rpoB -989R, 16S -786F / 16S -983R, 16S -412F / 16S -580R; 3 pairs of Corynebacterium-specific candidate primers: 16S -431F / 16S -563R, fusA -471F / fusA -645R, fusA -302F / fusA -533R; III. Third Stage: Verification of Specificity in PCR Experiments 1. The screening method in the third stage: The six candidate specific primers and ten bacterial universal primers obtained in the second stage were validated by routine PCR using 12 representative bacterial strains.

[0060] The 12 representative strains are as follows Corynebacterium kroppenstedtii DSM 44385 (CKC) Corynebacterium parakroppenstedtii MC-26 (CCTCC AB 2020210, CKC), Corynebacterium mucifaciens DSM 44903 (non-CKC corynebacterium) Corynebacterium glutamicum ATCC 13032 (non-CKC corynebacterium) Corynebacterium pekinense DSM 44726 (non-CKC corynebacterium) Corynebacterium amycolatum CIP 103452 (non-CKC corynebacterium)Mycobacterium abscessus ATCC 19977 (closely related genus) Mycobacterium bovis BCG Pasteur 1173P2 (closely related genus) Micrococcus luteus ATCC 4698 (distant bacteria) Pseudomonas aeruginosa ATCC 27853 (distant bacteria) Staphylococcus aureus ATCC 25923 (distant bacteria) Staphylococcus epidermidis ATCC 12228 (distant bacteria).

[0061] The experimental steps and conditions for routine PCR validation are described below: (1) Template preparation Fresh colonies of each representative strain (including CKC, non-CKC rod-shaped bacteria, closely related mycobacteria, and distantly related species) were inoculated into suitable liquid culture medium and cultured to the logarithmic growth phase.

[0062] Take the bacterial suspension, centrifuge at 12,000×g for 2 min, discard the supernatant, and resuspend in sterile, enzyme-free water.

[0063] Thermal lysis: Heat the bacterial culture in a 100 ℃ water bath for 10 min, cool it in an ice bath for 2 min, centrifuge briefly for 5 s, and take the supernatant as a routine PCR template.

[0064] (2) PCR reaction system Taking a 25 μL system as an example, the reaction components are: 2×Universal Green PCR Master Mix (Chengdu Rongwei Gene): 12.5 μL; Forward primer (10 μmol / L): 0.5 μL (final concentration 0.2 μmol / L); Reverse primer (10 μmol / L): 0.5 μL (final concentration 0.2 μmol / L); Template DNA (thermal lysis supernatant): 2.0 μL; Nuclease-free water: 9.5 μL; Total: 25 μL.

[0065] The primers included six pairs of candidate specific primers obtained in the second stage and ten pairs of universal bacterial primers.

[0066] (3) PCR amplification program Set the following cycling conditions on a standard gradient PCR instrument (refer to the MIQE guidelines and maintain consistency with subsequent qPCR conditions): Pre-denaturation (enzyme activation): 95 °C, 5 min, 1 cycle; Denaturation: 95 ℃, 30 s; Annealing: 55-60 ℃, 30 s (different primers were optimized according to the theoretical Tm, and most combinations were ultimately 60 ℃). Extension: 72 ℃, 30 s; Number of loops: 35; Final extension: 72 °C, 5 min, 1 cycle; 2. Results of the third stage of screening: (1) Of the 6 candidate specific primer pairs, 1 pair was eliminated due to the appearance of non-specific amplification bands, and 5 pairs were retained for qPCR comprehensive performance evaluation. The 5 retained primer pairs are as follows: 3 pairs of candidate CKC-specific primers: rpoB -703F / rpoB -989R, 16S-786F / 16S-983R, 16S-412F / 16S-580R; Two pairs of candidate genus-specific primers: 16S-431F / 16S-563R, fusA -471F / fusA -645R.

[0067] (2) All 10 pairs of universal primers were able to effectively amplify the expected bands in PCR and all entered the fourth stage for further screening.

[0068] IV. Fourth Stage: Comparison of Overall qPCR Performance 1. Screening of candidate specific primers (1) Screening method for candidate specific primers: Under the same DNA template, detection was performed according to the detection method of Example 1, wherein the primer sequences were adaptively adjusted to the 5 pairs of candidate specific primers retained in the third stage.

[0069] The DNA template corresponding to the CKC-specific primers is CKC strain 1 ( C. kroppenstedtii DNA template (DSM 44385).

[0070] The DNA template corresponding to the genus-specific primers was a non-CKC Corynebacterium strain. This non-CKC Corynebacterium strain was *Corynebacterium amyloliquefaciens*. Corynebacterium amycolatum CIP 103452, as a representative non-CKC Corynebacterium, was used for qPCR performance evaluation of Corynebacterium genus-specific primers.

[0071] Five pairs of candidate specific primers were evaluated by qPCR using the mean Cq value and the inter-batch coefficient of variation (CV%) as the main evaluation criteria. A lower Cq value (Quantification Cycle) indicates higher primer amplification efficiency, better sensitivity, and the ability to detect the target product in fewer cycles. A lower CV% (Coefficient of Variation) indicates better inter-batch reproducibility, higher stability, and suitability for long-term standardized use.

[0072] (2) Screening results of candidate specific primers The evaluation results of the five candidate specific primers are shown in Table 13. The results indicate that among the candidate specific primers, the candidate CKC specific primers... rpoB -703F / rpoB The -989R primer pair and the candidate genus-specific primer pair 16S-431F / 16S-563R primer pair had the lowest mean Cq and inter-batch coefficient of variation (CV%), demonstrating that these two pairs of candidate specific primers have high amplification efficiency, strong target recognition and amplification capabilities, good inter-batch repeatability, and strong stability and reliability.

[0073] Table 13 2. Screening of universal bacterial primers (1) Screening method for universal bacterial primers Ten universal primer pairs were used to evaluate the Cq values ​​of NTC background amplification in qPCR of three strains of bacteria (Corynebacterium kuribda DSM 44385, Corynebacterium amyloliquefaciens CIP103452, and Staphylococcus aureus ATCC 25923). The three primer pairs with the highest NTC Cq values ​​were selected and further verified by qPCR of GM pus sample (pus sample No. 20 in Example 2). The qPCR experimental methods and conditions were the same as in Example 1.

[0074] (2) Screening results of universal bacterial primers Non-specific amplification of NTC was observed in all 10 universal primer pairs, a common phenomenon with universal 16S rRNA primers (the literature reports an NTC Cq value of approximately 27, close to that of this invention). Higher NTC Cq values ​​indicate lower background noise and relatively better specificity of the universal primers. The three primer pairs with the highest NTC Cq values ​​were 357F / 518R, U16SRT-F / U16SRT-R, and V4-515F / V4-806R.

[0075] Among the three primer pairs, 357F / 518R exhibited the lowest Cq value in pus samples, indicating the best amplification efficiency and detection capability; it also showed the highest Cq value in NTC samples, indicating the lowest relative background amplification, and its melting curve was a single peak with no abnormal peaks. Considering these three performance characteristics, 357F / 518R performed optimally under the same clinical sample and NTC conditions, and was therefore selected as the universal primer combination.

[0076] Through experiments with GM pus samples, 357F / 518R was ultimately determined to be the optimal universal primer.

[0077] The cutoff value Cq=22.43 was objectively determined by ROC curve, which reduced the false positive rate of the universal primer to 3.8%. Compared with the prior art, the false positive rate of the present invention is at the lowest level. The comparison results are shown in Table 14.

[0078] Table 14 Note: Reference [1] Wu, YD; Shang, SQ; Li, JP; Yang, ZQ; Zheng, ZB; Du, LZ; Zhao, ZY [A broad-range 16S rRNA gene real-time PCR assay for the diagnosis of neonatal septicemia]. Zhonghua Er Ke Za Zhi 2007, 45 , 446-449; [2] Chen, L.-H.; Duan, Q.-J.; Cai, M.-T.; Wu, Y.-D.; Shang, S.-Q. RapidDiagnosis of Sepsis and Bacterial Meningitis in Children with Real-TimeFluorescent Quantitative Polymerase Chain Reaction Amplification in theBacterial 16S rRNA Gene. Clinical Pediatrics 2009, 48, 641-647, doi:10.1177 / 0009922809333972; [3] Ohlin, A.; Bäckman, A.; Björkqvist, M.; Mölling, P.; Jurstrand, M.; Schollin, J. Real-time PCR of the 16S-rRNA gene in thediagnosis of neonatal bacteraemia. Pediatric Journal 2008, 97 , 1376-1380, doi:10.1111 / j.1651-2227.2008.00924.x; [4] Zucol, F.; Ammann, RA; Berger, C.; Aebi, C.; Altwegg, M.; Niggli, FK; Nadal, D. Real-Time Quantitative Broad-Range PCR Assay for Detection of the 16S rRNA Gene Followed by Sequencing forSpecies Identification. Journal of Clinical Microbiology 2006, 44 , 2750-2759,doi:10.1128 / jcm.00112-06.

[0079] The above results indicate that, through multiple screening, this experiment yielded specific primer pairs and universal bacterial primer pairs with good specificity, amplification efficiency, and repeatability. Furthermore, the cutoff value Cq ≤ 22.43 was determined, resulting in an extremely low false positive rate.

[0080] Experiment Example 2: Evaluation of Detection Performance I. Experimental Methods Establishing the standard curve: C. kroppenstedtii DNA extract (6.26 ag–6.26 ng) and thermal lysis supernatant (1 × 10⁻⁶ g) from DSM 44385 0 ~1×10 9 The kits (CFU / mL) were prepared separately, with 9 dilution gradients for each gradient, 3 replicates per gradient, for a total of 9 runs. Detection was performed according to the kit and detection method in Example 1, with primer pairs as follows: rpoB -703F / rpoB -989R, 16S-431F / 16S-563R, 357F / 518R.

[0081] The lower limit of detection (LOD) refers to the lowest amount or concentration of DNA that can be detected with a specific amplification signal.

[0082] II. Experimental Results The detection results of the three primer pairs are shown in Table 15. When the DNA extract was used as a template, the LOD of both the CKC-specific primer and the genus-specific primer was 62.6 fg (approximately 1 × 10⁻⁶). 4 (CFU / mL) At the current detection limit, background amplification exists, and this detection limit is not applicable to general bacterial detection; when the thermal lysis supernatant is used as a template, the LOD of both CKC-specific primers and genus-specific primers is 1.0 × 10⁻⁶. 4 At CFU / mL, background amplification exists at the current detection limit, and this detection limit is not applicable to general bacterial detection.

[0083] Table 15 Note: CKCp indicates CKC-specific primers; CORp indicates Corynebacterium-specific primers; BACp indicates universal bacterial primers; all primers have undergone three technical replicates and three biological replicates; ND indicates not detected.

[0084] The statistical summary of the qPCR test results is shown in Table 16.

[0085] Table 16 The results show that the detection method of the present invention has a low detection limit and high sensitivity for detecting CKC and Corynebacterium spp., and the preferred primers of the present invention have high amplification efficiency; furthermore, it is shown that the amplification specificity of the primers can be confirmed by comparing the Tm values.

[0086] Experimental Example 3: Specificity Detection I. Experimental Methods Twelve representative bacterial strains were used to verify the specificity of the four preferred primer pairs. The four preferred primer pairs are those shown in SEQ ID NO. 1-8.

[0087] The 12 representative strains are as follows Corynebacterium kroppenstedtii DSM 44385 (CKC) Corynebacterium parakropenstedtii MC-26 (CCTCC AB 2020210, CKC), Corynebacterium mucilaginous DSM 44903 (non-CKC corynebacterium) Corynebacterium glutamicum ATCC 13032 (non-CKC corynebacterium) Corynebacterium pekinense DSM 44726 (non-CKC corynebacterium) Corynebacterium amycolatumCIP 103452 (non-CKC corynebacterium) Mycobacterium abscess ATCC 19977 (closely related genus) Mycobacterium bovis BCG Pasteur 1173P2 (closely related genus) Micrococcus luteus ATCC 4698 (distant bacteria) Pseudomonas aeruginosa ATCC 27853 (distant bacteria) Staphylococcus aureus ATCC 25923 (distant bacteria) Staphylococcus epidermidis ATCC 12228 (distant bacteria).

[0088] The qPCR reaction conditions were the same as in Example 1.

[0089] II. Experimental Results The qPCR test results are shown in Table 17 and Figure 2 As shown.

[0090] Table 17 The results showed that the CKC-specific primer (rpoB-703F / 989R) produced positive amplification only for CKC members (lanes 1 and 2), and was negative for all non-CKC Corynebacterium, closely related genera, and distantly related bacteria, with primer specificity reaching the CKC group level; the genus-specific primer (16S-431F / 563R) produced positive amplification only for Corynebacterium (lanes 1-6), and was negative for Mycobacterium and other distantly related bacteria, exhibiting good Corynebacterium specificity; the universal primer (357F / 518R) could effectively amplify all 12 bacterial strains, with background amplification at NTC (Cq≈28.2-28.3), a common phenomenon with universal 16S rRNA primers. This invention reduces the risk of false positives by using a Cq cutoff value (Cq≤22.43); the β-actin primer was negative for all bacteria, only amplifying human DNA, and can be used as an effective quality control for pus samples.

[0091] The above results indicate that the preferred primers of this invention have high specificity and can be used to specifically detect and determine CKC, non-CKC corynebacteria, corynebacteria spp., other bacterial infections, and non-bacterial infections.

[0092] As can be seen from the above embodiments and experimental examples, the present invention provides a multi-tube single-tube quantitative real-time PCR (qPCR) detection kit and its detection method for pathogen classification of GM pus specimens. The kit contains four pairs of primers, targeting: (1) group: CKC-specific target ( rpoB (2) Genus: Corynebacterium genus-specific target ( 16S rRNA (3) Universal: Bacterial universal targets (genes); (4) Universal targets: Bacterial universal targets (genes); (5) Universal targets: Bacterial universal targets (genes); (6) Universal targets: Bacterial universal targets (genes); (7) Universal targets: Bacterial universal targets (genes); (8) Universal targets: Bacterial universal targets (genes); (9) Universal targets: Bacterial universal targets (genes); (10) Universal targets: Bacterial universal targets (genes 16S rRNA (4) Human samples (gene common segments); β-actin (Genes). The hierarchical combination and interpretation of qPCR results enables the classification of GM pathogens. The detection method of this invention has high specificity, capable of distinguishing four GM types with a single test, exhibiting high detection rate and accuracy, short detection cycle, and high sensitivity. Furthermore, by setting a Cq cutoff value, it achieves an objective distinction between bacterial and non-bacterial infections, showing promising application prospects in the detection and treatment of granulomatous mastitis.

Claims

1. A PCR primer pair for the etiological classification of granulomatous mastitis, characterized in that, It includes: CKC-specific primer pairs and Corynebacterium-specific primer pairs; the nucleotide sequences of the CKC-specific primer pairs are shown in SEQ ID NO.1-2.

2. The PCR primer pair for etiological classification of granulomatous mastitis according to claim 1, characterized in that, The nucleotide sequences of the Corynebacterium-specific primer pairs are shown in SEQ ID NO.3-4.

3. The PCR primer pair for etiological classification of granulomatous mastitis according to claim 1, characterized in that, It also includes universal bacterial primer pairs and human housekeeping gene primer pairs.

4. The PCR primer pair for etiological classification of granulomatous mastitis according to claim 3, characterized in that: The nucleotide sequences of the bacterial universal primer pairs are shown in SEQ ID NO.5-6.

5. The PCR primer pair for etiological classification of granulomatous mastitis according to claim 3, characterized in that: Human housekeeping genes include β-actin, GAPDH, and 18S rRNA. The nucleotide sequences of the β-actin primer pair are shown in SEQ ID NO. 7-8.

6. Use of the PCR primer pair for etiological classification of granulomatous mastitis as described in any one of claims 1-5 in the preparation of a kit for etiological classification of granulomatous mastitis.

7. The use of the PCR primer pair for etiological classification of granulomatous mastitis according to claim 6 in the preparation of a kit for etiological classification and detection of granulomatous mastitis, characterized in that, The etiological classification includes: CKC infection and non-CKC rod-shaped bacillus infection.

8. The use of the PCR primer pair for etiological classification of granulomatous mastitis according to claim 7 in the preparation of a kit for etiological classification and detection of granulomatous mastitis, characterized in that, The basis for the etiological classification includes: performing PCR amplification reactions on the test samples using PCR primers; When the CKC-specific primer pair produces a specific amplification product, it is determined to be CKC positive; when the CKC-specific primer pair produces no specific amplification product, but the Corynebacterium genus-specific primer pair produces a specific amplification product, it is determined to be non-CKC Corynebacterium positive. The PCR amplification reaction conditions included: denaturation at 95℃ for 10 seconds, annealing at 55-65℃ for 30 seconds, extension at 72℃ for 30 seconds, and 40 cycles.

9. The use of the PCR primer pair for etiological classification of granulomatous mastitis according to claim 8 in the preparation of a kit for etiological classification and detection of granulomatous mastitis, characterized in that, The etiological classification also includes: bacterial infections other than Corynebacterium and non-bacterial infections; The criteria for pathogen classification also include: when the quantitative cycle number Cq of the PCR amplification reaction using the universal bacterial primer pair is ≤22.43, and when the CKC-specific primer pair and the Corynebacterium genus-specific primer pair produce no specific amplification products, the result is determined to be positive for bacteria other than Corynebacterium. When the quantitative cycle number Cq of the PCR amplification reaction using the universal bacterial primer pair is greater than 22.43, and the reaction of the CKC specific primer pair and the Corynebacterium genus specific primer pair produces no specific amplification product, the result is determined to be bacterial negative. And / or, the kit may also include amplification reagents, positive controls, and negative controls.

10. A reagent kit, characterized in that: It comprises the PCR primer pair for etiological classification of granulomatous mastitis as described in any one of claims 1-5.

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