Fluorescent quantitative PCR (Polymerase Chain Reaction) detection method of lactobacillus plantarum
By designing specific primers and combining them with TaqMan probes, and optimizing the PCR reaction system, the problems of long detection time, high false positive rate, and poor matrix compatibility of Lactobacillus plantarum were solved, achieving rapid, accurate, and highly sensitive detection suitable for complex samples.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-04
- Publication Date
- 2026-03-31
AI Technical Summary
Existing detection methods for Lactobacillus plantarum are time-consuming, prone to false positives, have low sensitivity, and poor matrix compatibility, making it difficult to achieve rapid, accurate, and highly sensitive detection, especially in complex samples.
By designing specific primers and combining them with TaqMan probes, along with an optimized PCR reaction system, conserved regions are identified through multiple sequence alignment, and sample pretreatment and nucleic acid extraction are optimized to achieve detection with high specificity and high sensitivity.
It achieves detection within 2 hours, has high specificity and high sensitivity, and can accurately detect low concentrations of Lactobacillus plantarum in complex samples. It is suitable for a variety of practical samples such as water bodies and fermented foods, meeting the needs of rapid monitoring and industrial quality control.
Smart Images
Figure CN121759620A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biochemical detection technology, specifically relating to a fluorescence quantitative PCR detection method for Lactobacillus plantarum. Background Technology
[0002] Lactobacillus plantarum, as an important probiotic, is widely used in industries such as aquaculture, food fermentation, and health products. Rapid and accurate quantitative detection of this bacteria is crucial for ensuring product quality and safety. Currently, traditional isolation and culture methods remain the commonly used detection approach. This method requires anaerobic culture for over 72 hours, followed by identification and counting based on colony morphology and biochemical characteristics. The entire process is cumbersome and time-consuming, failing to meet the urgent need for rapid on-site monitoring.
[0003] With the development of molecular biology techniques, quantitative real-time PCR (qPCR) has become an important tool for microbial detection. Among these methods, the SYBR Green dye method is widely used for detecting *Lactobacillus plantarum* due to its ease of operation. However, this method is susceptible to interference from non-specific amplification products and primer dimers, leading to a high risk of false positives and affecting detection accuracy. On the other hand, while the TaqMan probe method, with its higher specificity, theoretically holds great potential, current probe detection systems for *Lactobacillus plantarum* are still imperfect, generally suffering from insufficient detection sensitivity and poorly optimized primer-probe combinations, making it difficult to effectively detect low concentrations of target bacteria in complex sample matrices.
[0004] Furthermore, existing detection methods generally exhibit poor adaptability to complex real-world samples such as water bodies and fermented foods. Interfering substances in the samples can severely inhibit PCR reactions, leading to detection failures or inaccurate quantification. Currently, there is a lack of a highly sensitive, standardized detection protocol that can effectively overcome matrix inhibition and achieve accurate quantification across sample types. Therefore, developing a rapid detection technology for *Lactobacillus plantarum* that combines high specificity, high sensitivity, and broad matrix adaptability is particularly necessary. Summary of the Invention
[0005] This invention provides a fluorescence quantitative PCR detection method for Lactobacillus plantarum, which overcomes the shortcomings of existing Lactobacillus plantarum detection methods, such as excessive time consumption, easy generation of false positives, low sensitivity, and poor matrix adaptability. It provides a detection method that can achieve rapid, accurate, highly sensitive detection and is applicable to complex matrices.
[0006] On the one hand, the present invention provides a real-time quantitative PCR detection method for Lactobacillus plantarum, which adopts the following technical solution: A real-time quantitative PCR detection method for Lactobacillus plantarum includes the following steps: S1. Log in to the NCBI GenBank database, download the full sequence of conserved genes of Lactobacillus plantarum and the homologous gene sequences of non-target bacteria, identify the conserved regions unique to Lactobacillus plantarum through multiple sequence alignment, design candidate primers and TaqMan probes and synthesize them. S2. Select a standard strain of Lactobacillus plantarum, inoculate it into liquid culture medium and culture it to the logarithmic phase, then extract genomic DNA after centrifugation; S3. Add to the Q-PCR reaction system, amplify, and screen to obtain primer LP3; S4. Select standard strains of Lactobacillus plantarum, inoculate them into liquid culture medium for anaerobic culture, perform serial dilution, centrifuge and extract genomic DNA. Use the DNA of each concentration of strain as a template, use LP3 primer and probe combination, add to the Q-PCR reaction system for amplification, and plot a standard curve. S5. Collect samples and extract DNA, perform Q-PCR reaction, and calculate the concentration of lactobacillus by comparing with the standard curve.
[0007] Preferably, the candidate primers in step S1 include three pairs of primers: LP1, LP2, and LP3, wherein: The sequence of the LP1 forward primer is SEQ ID NO.1; The sequence of the LP1 reverse primer is SEQ ID NO.2; The sequence of the LP2 forward primer is SEQ ID NO.3; The sequence of the LP2 reverse primer is SEQ ID NO.4; The sequence of the LP3 forward primer is SEQ ID NO.5; The sequence of the LP3 reverse primer is SEQ ID NO.6.
[0008] Preferably, the TaqMan probe includes a fluorescent reporter group, a specific recognition sequence LP-P, and a fluorescence quenching group; The fluorescent reporter group is FAM; The specific recognition sequence LP-P is SEQ ID NO.7; The fluorescence quenching group is BHQ-1.
[0009] Preferably, the Q-PCR reaction system includes 2×5G enzyme, forward primer, reverse primer, TaqMan probe, DNA template, and deionized water.
[0010] Preferably, the concentration range of the forward and reverse primers in the reaction system is 0.2~0.4 μmol / L, with the optimal concentration being 0.3 μmol / L.
[0011] Preferably, the concentration of the TaqMan probe in the reaction system is in the range of 0.08~0.12 μmol / L, with the optimum being 0.1 μmol / L.
[0012] Preferred non-target bacteria include Aeromonas versicolor ( Aeromonas veronii ), Escherichia coli ( Escherichia coli Acinetobacter ( ) Acinetobacter sp.), Shigella sigmatis ( Plesiomonas shigelloides Lactobacillus casei ( Lactobacillus casei Shewanella ( Shewanella sp.).
[0013] Preferably, the method used to extract genomic DNA is the metal bath method.
[0014] In summary, the beneficial effects of the present invention are as follows: This invention achieves high-performance detection of *Lactobacillus plantarum* by providing a set of specifically designed primers and TaqMan probes, combined with an optimized detection system. Compared with existing technologies, its most significant advantage lies in its superior detection performance: the method possesses high specificity, effectively avoiding cross-reactivity with non-target bacteria and false positives; it also exhibits high sensitivity, with a detection limit as low as 3.5 × 10⁻⁶. 2 With a concentration of cfu / mL, it can accurately capture low concentrations of target bacteria in complex samples.
[0015] Furthermore, this invention demonstrates significant advantages in practicality and efficiency. The entire detection process can be completed within 2 hours, achieving rapid quantitative analysis compared to traditional culture methods that take several days. By optimizing sample pretreatment and nucleic acid extraction protocols, this invention also exhibits broad matrix adaptability, enabling stable and accurate detection of various complex real-world samples such as water bodies and fermented foods, meeting the urgent needs of rapid on-site monitoring and industrial quality control. Attached Figure Description
[0016] Figure 1 The results of real-time PCR screening of candidate primer-probe combinations are shown in the figure. Figure 2 The image shows the results of real-time quantitative PCR for specific detection. Figure 3 The image shows the results of quantitative real-time PCR for sensitivity testing. Figure 4 This is a standard curve for the detection of Lactobacillus plantarum. Detailed Implementation
[0017] The present invention will be further described in detail below with reference to the embodiments.
[0018] Example Example 1 The specific steps for primer pair and probe design, screening, and validation are as follows: S1: Log in to the NCBI GenBank database and download 10 strains of Lactobacillus plantarum ( Lactobacillus plantarum The conserved full-length sequences of the target gene were obtained, and the homologous gene sequences of six non-target bacteria (such as Aeromonas verrucosum, Lactobacillus plantarum, and Lactobacillus casei) were downloaded as controls. Multiple sequence alignment was performed using DNAMAN 9.0 software to identify conserved regions specific to Lactobacillus plantarum as detection targets.
[0019] S2. Using Primer 3.0 software, three pairs of candidate primers (LP1~LP3) and one TaqMan probe were designed for the above-mentioned conserved region. All primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and their sequence information is shown in Table 1.
[0020] Table 1 Primer and probe sequences
[0021] S3. Select a standard strain of *Lactobacillus plantarum*, inoculate it into MRS liquid medium, and culture it anaerobically with shaking at 36℃±1℃ until the logarithmic growth phase (OD50). 600 ≈0.6). Take 1 mL of bacterial culture, centrifuge at 8000 r / min for 10 min to collect the bacterial cells, and extract genomic DNA using the DNA / RNA co-extraction and purification kit (metal bath method) provided by Jiangsu Liezhen Biotechnology Co., Ltd., and store at -20℃ for later use.
[0022] S4. Q-PCR used a 20 μL reaction system: 10 μL of 2×5G enzyme, 0.6 μL each of 10 μmol / L forward and reverse primers, 0.2 μL of 10 μmol / L probe, 3.0 μL of DNA template, and 5.6 μL of deionized water. Amplification program: 95℃ pre-denaturation for 2 min; 40 cycles (95℃ denaturation for 10 s, 58℃ annealing and extension for 30 s, fluorescence signal collection stage).
[0023] Reference Figure 1 The x-axis represents the PCR cycle number, and the y-axis represents the fluorescence signal intensity. The curves in the figure represent three combinations of primers and probes. Among them, the LP3 primer pair + LP-P probe combination (black curve) has the earliest fluorescence signal peak and the smallest Ct value, and the curve is a typical "S" shape with an average Ct value of 18.19±0.3. The fluorescence curve has a high peak and a stable baseline. The other combinations have weak fluorescence signals or no amplification, indicating that this combination is the optimal screening result.
[0024] Example 2: The specific steps for verifying the specificity of the optimal primer-probe combination for Lactobacillus plantarum are as follows: S1. Select one target bacterium (Lactobacillus plantarum) and six common non-target bacteria, all of which are cultured to the logarithmic phase. Use the instruction manual of the DNA / RNA co-extraction and purification kit (metal bath method) provided by Jiangsu Liezhen Biotechnology Co., Ltd. to extract genomic DNA. The strain information is shown in Table 2.
[0025] Table 2 List of specific test strains
[0026] S2 and Q-PCR followed the steps in S4 of Example 1, using the LP3 primer pair + LP-P probe combination, with a negative control (DEPC water), and each sample was replicated 3 times.
[0027] Reference Figure 2 Only *Lactobacillus plantarum* showed specific amplification (Ct value 15.1±0.2, typical "S" curve), while no amplification signal was observed in the six non-target bacteria and the negative control, indicating that the combination had good specificity and no risk of cross-amplification.
[0028] Example 3: The linear range and limit of detection of the LP3 primer-probe combination were determined based on the LP3 primer-probe combination, and a standard curve was established. The specific steps are as follows: S1. Prepare pure culture of *Lactobacillus plantarum*, and determine the initial concentration as 3.5 × 10⁻⁶ using the MRS plate count method (anaerobic culture at 36℃ for 48 h). 8 cfu / mL; 7 concentration gradients were obtained by serially diluting 10-fold with sterile physiological saline: 3.5 × 10⁻⁶ cfu / mL. 7 3.5×10 6 3.5×10 5 3.5×10 4 3.5×10 3 3.5×10 2 3.5×10 1 cfu / mL.
[0029] S2. Take 1 mL of bacterial culture of each concentration, centrifuge at 8000 r / min for 10 min, resuspend the precipitate in sterile physiological saline, and extract DNA using the method in step S1 of Example 2.
[0030] S3. Q-PCR detection was performed using DNA of various concentrations as templates. The LP3 primer pair + LP-P probe combination was used, and the detection was performed according to the system and procedure of Example 1. Each concentration was repeated 3 times.
[0031] S4. Plot a standard curve with the logarithm of bacterial concentration (lg Cfu / mL) on the x-axis and the average Ct value on the y-axis. Reference Figure 3The curves in the figure correspond to different concentrations of *Lactobacillus plantarum* bacterial solutions (3.5 × 10¹ ~ 3.5 × 10¹). 7 (cfu / mL), concentration ≥3.5×10 2 The curves for CFU / mL all showed an "S" shape, with concentrations < 3.5 × 10⁻⁶. 2 The curve CT value of cfu / mL is <35, the amplification efficiency is 102%, which meets the standard of real-time PCR and visually demonstrates the lowest detection limit of the method.
[0032] Reference Figure 4 The standard curve equation is y = -3.3154x + 41.413, with a correlation coefficient R² = 0.99, indicating that the correlation coefficient is within the range of 3.5 × 10² to 3.5 × 10⁻⁶. 7 Within the cfu / mL concentration range, the bacterial concentration and Ct value show a good linear relationship, which can be used for accurate quantification of Lactobacillus plantarum in samples.
[0033] Example 4: The specific steps for detecting the concentration of Lactobacillus plantarum in actual complex samples are as follows: S1. Collect three types of actual samples: ① freshwater fishpond water; ② fermented yogurt; ③ kimchi juice. Each sample was tested in triplicate. Water samples were filtered through a membrane and then centrifuged. Food samples were centrifuged directly at 8000 r / min for 10 min, and the precipitate was resuspended in sterile physiological saline. DNA was extracted using the method described in step S1 of Example 2.
[0034] S2 and Q-PCR followed step S4 in Example 1, using the LP3 primer pair + LP-P probe combination. The concentration of *Lactobacillus plantarum* in the sample was calculated using the standard curve from Example 3, and the results are shown in Table 3. The LP3 primer and probe combination is suitable for water samples with a sampling volume of 5 mL or more (to avoid missed detections at low concentrations) and can effectively remove interference from proteins and oils in fermented foods, ensuring accurate quantification.
[0035] Table 3. Detection of Lactobacillus plantarum concentration in different samples
[0036] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.
Claims
1. A method for detecting Lactobacillus plantarum using real-time quantitative PCR, characterized in that, Includes the following steps: S1. Log in to the NCBI GenBank database, download the full sequence of conserved genes of Lactobacillus plantarum and the homologous gene sequences of non-target bacteria, identify the conserved regions unique to Lactobacillus plantarum through multiple sequence alignment, design candidate primers and TaqMan probes and synthesize them. S2. Select a standard strain of Lactobacillus plantarum, inoculate it into liquid culture medium and culture it to the logarithmic phase, then extract genomic DNA after centrifugation; S3. Add to the Q-PCR reaction system, amplify, and screen to obtain primer LP3; S4. Select standard strains of Lactobacillus plantarum, inoculate them into liquid culture medium for anaerobic culture, perform serial dilution, centrifuge and extract genomic DNA. Use the DNA of each concentration of strain as a template, use LP3 primer and probe combination, add to the Q-PCR reaction system for amplification, and plot a standard curve. S5. Collect samples and extract DNA, perform Q-PCR reaction, and calculate the concentration of lactobacillus by comparing with the standard curve obtained in step S4.
2. The method for detecting *Lactobacillus plantarum* using real-time PCR according to claim 1, characterized in that, The candidate primers in step S1 include LP1, LP2 and LP3; LP1 includes LP1-F and LP1-R; The sequence of LP1-F is SEQ ID NO.1; The sequence of LP1-R is SEQ ID NO.2; LP2 includes LP2-F and LP2-R; The sequence of LP2-F is SEQ ID NO.3; The sequence of LP2-R is SEQ ID NO.4; LP3 includes LP3-F and LP3-R; The sequence of LP3-F is SEQ ID NO.5; The sequence of LP3-R is SEQ ID NO.
6.
3. The method for detecting *Lactobacillus plantarum* using real-time PCR according to claim 1, characterized in that, In step S1, the TaqMan probe includes a fluorescent reporter group, a specific recognition sequence LP-P, and a fluorescent quenching group. The fluorescent reporter group is FAM; The specific recognition sequence LP-P is SEQ ID NO.7; The fluorescence quenching group is BHQ-1.
4. The method for detecting Lactobacillus plantarum using real-time PCR according to claim 1, characterized in that, The Q-PCR reaction system includes 2×5G enzyme, forward primer, reverse primer, TaqMan probe, DNA template, and deionized water.
5. The method for detecting *Lactobacillus plantarum* using real-time PCR according to claim 4, characterized in that, The concentration range of the forward and reverse primers in the reaction system is 0.2~0.4 μmol / L.
6. The method for detecting Lactobacillus plantarum using real-time PCR according to claim 4, characterized in that, The concentration of the TaqMan probe in the reaction system ranges from 0.08 to 0.12 μmol / L.
7. The method for detecting Lactobacillus plantarum using real-time PCR according to claim 4, characterized in that, The non-target bacteria include Aeromonas vera, Escherichia coli, Acinetobacter bacillus, Shigella-like bacteria, Lactobacillus casei, and Shewanella.
8. The method for detecting Lactobacillus plantarum using real-time PCR according to claim 4, characterized in that, The method used to extract genomic DNA is the metal bath method.