Method for detecting plasmid copy number in engineering bacteria by qPCR (quantitative polymerase chain reaction)

Abstract

The invention relates to the field of biological medicine, in particular to a method for detecting plasmid copy number in engineering bacteria by qPCR (quantitative polymerase chain reaction). The method comprises the following steps: constructing standard plasmids, establishing methodology, carrying out methodology verification on the method, and carrying out plasmid copy number detection on the strain by using the method. The standard plasmid comprises a reference gene alaA and to-be-detected genes bla and Kan, the ratio of the reference gene alaA to the to-be-detected genes bla to Kan is 1: 1: 1, and the standard plasmid can be used for detecting the copy number of engineered plasmids containing ampicillin resistance genes or kanamycin resistance genes in engineered strains.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a method for detecting plasmid copy number in engineered bacteria using qPCR. Background Technology

[0002] Plasmid copy number refers to the average number of plasmid DNA molecules per chromosome in a bacterial cell, and is a key indicator for measuring the core characteristics of engineered bacteria. Based on its numerical value, it can be divided into three main categories: plasmids with a copy number in the range of 1 to 10 are defined as low copy number plasmids; those in the range of 11 to 20 are classified as medium copy number plasmids; and those with a copy number exceeding 20 are identified as high copy number plasmids.

[0003] In terms of gene composition, both the β-lactamase gene (bla) and the kanamycin resistance gene (Kan) on the plasmid are single-copy genes, while the alaA gene is a typical single-copy gene on the chromosomal DNA of *E. coli*. Based on this gene copying characteristic, in actual detection, for plasmids carrying ampicillin resistance, the copy number of the plasmid can be calculated by determining the copy ratio of the bla gene to the alaA gene; for plasmids carrying kanamycin resistance, the copy number can be accurately determined by using the copy ratio of the Kan gene to the alaA gene.

[0004] The commonly used detection methods are generally divided into the following three types: (1) Ultraviolet spectrophotometry: Although the required equipment is simple and the cost is low, the steps are complicated, the DNA extraction and gel recovery efficiency varies greatly, it is impossible to distinguish between supercoiled and open circular plasmids, and it requires knowledge of the precise size of the host chromosome DNA, resulting in very inaccurate detection results. (2) Real-time quantitative PCR: This is one of the most commonly used and reliable methods, with high sensitivity and high specificity. (3) Digital PCR: It can perform absolute quantification, does not depend on the standard curve, and has the highest precision and accuracy. However, the disadvantage is that the cost of instruments and reagents is very high, and although the throughput is high, the single run time is long.

[0005] While a method for detecting plasmid copy number in *E. coli* using real-time quantitative PCR exists (see "A Method for Detecting Plasmid Copy Number in *E. coli*"), it lacks standardized plasmid construction. It calculates the target gene copy number solely based on the relative difference in Ct values ​​(threshold cycle number), without verifying the amplification efficiency of the two primer pairs. For relative quantification, the core prerequisite is that the amplification efficiencies of the target gene and the internal reference gene must be close to 100%. Inconsistent efficiencies will result in significant errors. Summary of the Invention

[0006] In view of this, the present invention selects real-time quantitative PCR, which is relatively simple to operate and has accurate detection, as the basic method of this method, and provides a method for detecting the copy number of plasmids in engineered bacteria using qPCR. At the same time, a standard plasmid is constructed, which contains the internal reference gene alaA and the target genes bla and Kan, and the ratio of the three is 1:1:1. It can be used to accurately detect the copy number of engineered plasmids containing ampicillin resistance genes or kanamycin resistance genes in engineered bacterial strains.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a standard quality plasmid containing an internal reference gene, a first test gene, and a second test gene with the same copy number;

[0009] The internal reference gene is a single-copy gene of engineered bacteria;

[0010] The first and second genes to be tested are marker genes.

[0011] In some specific embodiments of the present invention, the backbone of the standard quality plasmid is pGEX-6p-2, the internal reference gene is alaA, the first gene to be tested is bla, and the second gene to be tested is Kan.

[0012] In some specific embodiments of the present invention, the insertion site of the alaA of the above-mentioned standard quality grain in pGEX-6p-2 is between BamH Ⅰ and EcoR Ⅰ;

[0013] The insertion site of Kan in pGEX-6p-2 is between Not I and Sal I.

[0014] In some specific embodiments of the present invention, the method for preparing the above-mentioned standard quality particles includes:

[0015] Insert alaA between BamH Ⅰ and EcoR Ⅰ on pGEX-6p-2;

[0016] Kan is inserted between Not I and Sal I on pGEX-6p-2, so that the copy number of alaA, Kan and bla on pGEX-6p-2 is 1:1:1, thus obtaining the standard quality grain.

[0017] This invention also provides the application of the above-mentioned standard plasmid in detecting the plasmid copy number of engineered bacteria.

[0018] In some specific embodiments of the present invention, the above application ensures the accuracy of the plasmid copy number based on the standard plasmid.

[0019] In some specific embodiments of the present invention, ensuring the accuracy of the plasmid copy number in the above application includes:

[0020] To ensure the accuracy of plasmid copy number of the engineered bacteria under test, at least one of the following should be compared to determine the differences in results between primer / probe sets or reaction conditions, based on amplification using a standard plasmid as a template:

[0021] a) Amplification results of the internal reference gene and the amplification results of the first gene to be tested;

[0022] b) Amplification results of the internal reference gene and the amplification results of the second gene to be tested;

[0023] The reaction conditions include the reaction system and / or the reaction procedure;

[0024] The parameters of the reaction program include temperature, time, and number of cycles;

[0025] The amplification results may be presented in forms including but not limited to Ct value, copy number, mass concentration, and molar concentration.

[0026] This invention also provides a method for detecting plasmid copy number in engineered bacteria based on qPCR, comprising:

[0027] S1: Use standard quality particles to verify the amplification efficiency of the internal reference gene primer and probe set, the first test gene primer and probe set, and the second test gene primer and probe set.

[0028] S2: Using the internal reference gene primer and probe set, the internal reference gene of the engineered bacteria to be tested is amplified to obtain the Ct value of the internal reference gene;

[0029] S3: Using the first test gene primer and probe set as a template, the first test gene on the plasmid of the engineered bacteria is amplified to obtain the Ct value of the first test gene;

[0030] S4: Using the second test gene primer and probe set as a template, the second test gene on the plasmid of the engineered bacteria is amplified to obtain the Ct value of the second test gene;

[0031] S5: Calculate the plasmid copy number based on at least one of the following:

[0032] a) The Ct value of the internal reference gene and the Ct value of the first gene to be tested;

[0033] b) The Ct value of the internal reference gene and the Ct value of the second gene to be tested;

[0034] The standard quality grain contains the internal reference gene, the first gene to be tested, and the second gene to be tested, all in equal copy numbers.

[0035] In some specific embodiments of the present invention, in S1 of the above method, the amplification efficiency of the internal reference gene primer probe set, the first test gene primer probe set, and the second test gene primer probe set are all between 83.30% and 110.17%, and can be 83%, 84%, 85%, 86%, 86.32%, 87%, 88%, 89%, 89.57%, 90%, 91%, 92%, 93%, 93.07%, 94%, 95%, 96%, 96.84%, 97%, 98%, 99%, 100%, 100.92%, 101%, 102%, 103%, 104%, 105%, 105.35%, 106%, 107%, 108%, 109%, or 110%.

[0036] In some specific embodiments of the present invention, after S1 and before S2 of the above method, the method further includes:

[0037] If the amplification efficiency difference exceeds the acceptable range, at least one of the internal reference gene primer and probe set, the first gene to be tested primer and probe set, and the second gene to be tested primer and probe set shall be changed; otherwise, proceed to the next step.

[0038] The acceptable range refers to the amplification efficiency of the internal reference gene primer and probe set, the first test gene primer and probe set, and the second test gene primer and probe set all being between 83.30% and 110.17%, which can be 83%, 84%, 85%, 86%, 86.32%, 87%, 88%, 89%, 89.57%, 90%, 91%, 92%, 93%, 93.07%, 94%, 95%, 96%, 96.84%, 97%, 98%, 99%, 100%, 100.92%, 101%, 102%, 103%, 104%, 105%, 105.35%, 106%, 107%, 108%, 109%, or 110%.

[0039] In some specific embodiments of the present invention, after S2 and before S3 of the above method, the method further includes:

[0040] Verify the accuracy of plasmid copy numbers obtained based on the internal reference gene primer-probe set, the first test gene primer-probe set, and the second test gene primer-probe set using any of the following methods. If the accuracy exceeds the acceptable range, change at least one of the internal reference gene primer-probe set, the first test gene primer-probe set, and the second test gene primer-probe set; otherwise, proceed to the next step:

[0041] i) Determine the copy number of the plasmid;

[0042] ii) Engineered bacteria containing plasmids with a defined copy number;

[0043] The acceptable range refers to the amplification efficiency of the internal reference gene primer and probe set, the first test gene primer and probe set, and the second test gene primer and probe set all being between 83.30% and 110.17%, which can be 83%, 84%, 85%, 86%, 86.32%, 87%, 88%, 89%, 89.57%, 90%, 91%, 92%, 93%, 93.07%, 94%, 95%, 96%, 96.84%, 97%, 98%, 99%, 100%, 100.92%, 101%, 102%, 103%, 104%, 105%, 105.35%, 106%, 107%, 108%, 109%, or 110%.

[0044] In some specific embodiments of the present invention, after S2 and before S3 of the above method, the method further includes:

[0045] Verify the accuracy of the plasmid copy number obtained based on the first, second, and third reaction conditions using any of the following methods. If the accuracy exceeds the acceptable range, change at least one of the first, second, and third reaction conditions; otherwise, proceed to the next step:

[0046] i) Determine the copy number of the plasmid;

[0047] ii) Engineered bacteria containing plasmids with a defined copy number;

[0048] The primer and probe set for the first reaction condition is the primer and probe set for the internal reference gene;

[0049] The primer and probe set for the second reaction condition is the primer and probe set for the first gene to be detected;

[0050] The primer and probe set for the third reaction condition is the primer and probe set for the second gene to be tested;

[0051] The acceptable range refers to:

[0052] a) The amplification efficiencies of the first reaction condition, the second reaction condition, and the third reaction condition are all between 83.30% and 110.17%, and can be 83%, 84%, 85%, 86%, 86.32%, 87%, 88%, 89%, 89.57%, 90%, 91%, 92%, 93%, 93.07%, 94%, 95%, 96%, 96.84%, 97%, 98%, 99%, 100%, 100.92%, 101%, 102%, 103%, 104%, 105%, 105.35%, 106%, 107%, 108%, 109%, or 110%.

[0053] In some specific embodiments of the present invention, the reaction conditions of the above method include the reaction system and / or the reaction procedure;

[0054] The reaction system includes the components and proportions required for the amplification reaction;

[0055] The parameters of the reaction program include temperature, time, and number of cycles.

[0056] In some specific embodiments of the present invention, the internal reference gene in the above method is a single-copy gene of engineered bacteria;

[0057] The first and second genes to be tested are marker genes.

[0058] In some specific embodiments of the present invention, the internal reference gene of the above method is alaA, the first gene to be tested is bla, and the second gene to be tested is Kan.

[0059] In some specific embodiments of the present invention, the skeleton of the standard quality grain of the above method is pGEX-6p-2.

[0060] In some specific embodiments of the present invention, the plasmid copy number of the above-described application or method is obtained based on a standard curve;

[0061] The standard curve is prepared by qPCR detection of a series of gradients of known amounts of nucleic acid molecules or plasmids;

[0062] The quantities mentioned include, but are not limited to, mass, amount of substance, and copy number.

[0063] In some specific embodiments of the present invention, the conversion relationship between the internal reference gene, the first gene to be tested, the second gene to be tested, and the plasmid copy number in the above-described application or method is calculated based on the DNA quality and DNA length of the internal reference gene, the first gene to be tested, and the second gene to be tested, as shown in the formula:

[0064] .

[0065] The standard plasmids of this invention can be used to quantitatively analyze the copy number of engineered plasmids containing ampicillin (or kanamycin) resistance genes in engineered bacterial strains. This overcomes the limitation of the methods described in the prior art, which can only detect plasmid copy numbers in engineered plasmid strains containing kanamycin resistance genes.

[0066] qPCR amplification of the standard plasmid showed amplification efficiencies of 83.30% to 110.17% for the internal reference gene alaA and the target genes bla and Kan, with recoveries ranging from 70% to 130%, demonstrating that the standard contained an internal reference gene alaA: target gene bla: target gene Kan ratio of 1:1:1. Simultaneously, using a bacterial strain containing an engineered plasmid (carrying an ampicillin resistance gene) as an example, the method was validated for its limit of quantitation, range, linearity, accuracy, intermediate precision, robustness, and specificity. Finally, the plasmid copy number of a bacterial strain containing an engineered plasmid (carrying an ampicillin resistance gene) was determined using this method.

[0067] The establishment of this method can provide technical support for the accurate assessment of plasmid stability and target gene expression levels in engineered strains, and further provide a scientific and reliable theoretical basis and data reference for the quality control of subsequent related research and industrial production processes. Attached Figure Description

[0068] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0069] Figure 1 The standard quality particle pAK-001 spectrum is shown;

[0070] Figure 2 The amplification diagram of the standard quality grains is shown. Detailed Implementation

[0071] This invention discloses a method for detecting plasmid copy number in engineered bacteria using qPCR. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the same result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0072] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0073] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0074] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0075] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0076] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0077] This invention discloses a method for detecting plasmid copy number in engineered bacteria using qPCR. In one implementation, the method includes:

[0078] (1) Construction of standard plasmid: The plasmid contains a single copy of the gene alaA on the Escherichia coli chromosomal DNA, a single copy of the β-lactamase gene (bla) on the plasmid, and the kanamycin resistance gene (Kan).

[0079] (2) Methodology establishment: Design specific primers and probes, amplify standard plasmids using qPCR technology, plot standard curves, and verify the primer amplification efficiency of the above three genes;

[0080] (3) Taking a bacterial strain containing an engineered plasmid (carrying an ampicillin resistance gene) as an example, the methodological verification of this method was carried out;

[0081] (4) The plasmid copy number of a bacterial strain containing an engineered plasmid (carrying an ampicillin resistance gene) was detected using this method.

[0082] In specific implementation, the standard plasmid contains the internal reference gene alaA and the test genes bla and Kan, with a ratio of 1:1:1. It can be used to detect the copy number of engineered plasmids containing ampicillin resistance genes or kanamycin resistance genes in engineered bacterial strains.

[0083] In one example, the specific process for constructing the aforementioned universal standard plasmid includes: firstly, synthesizing the typical single-copy gene alaA from the E. coli chromosomal DNA into the pGEX-6p-2 vector to obtain the pGEX-6p-2-alaA vector; then, amplifying the Kan gene on the pET-28a plasmid using PCR technology, and performing double enzyme digestion on the amplified Kan gene and the pGEX-6p-2-alaA vector; then, after ligation, introducing it into the XL1 strain; and finally, obtaining the target standard plasmid through strain amplification culture and plasmid extraction.

[0084] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0085] The present invention will be further illustrated below with reference to the embodiments.

[0086] Example 1: Construction of standard pAK-001 plasmid

[0087] 1. Experimental Methods

[0088] First, a third-party company cloned the typical single-copy gene alaA from the *E. coli* chromosomal DNA into the pGEX-6p-2 vector. BamH I and EcoR I were selected as double-enzyme digestion sites to construct the recombinant vector pGEX-6p-2-alaA. Subsequently, using the pET-28a plasmid as a template, the Kan gene was amplified by PCR (primer sequences are detailed in Table 1). The PCR reaction conditions were set as follows: 98℃ pre-denaturation for 4 minutes; followed by a cycling phase of 30 cycles, each cycle consisting of 98℃ denaturation for 40 seconds, 62℃ annealing for 1 minute, and 72℃ extension for 1 minute; after cycling, the final extension was completed at 72℃ for 7 minutes. The PCR-amplified Kan gene fragment and the pGEX-6p-2-alaA vector were then subjected to Not I and Sal I double digestion, respectively. The digestion products were ligated to construct the recombinant plasmid, which was then transformed into XL1 competent strains. The transformed strain was cultured to expand its scale, and the target standard plasmid was obtained through plasmid extraction. The obtained target standard plasmid was then sent to a third-party company for gene sequencing verification. The sequencing results were consistent with the expected sequence, indicating that the pAK-001 standard plasmid was successfully constructed.

[0089] Table 1 Primers for Kan-increasing enzyme cleavage sites

[0090]

[0091] 2. Experimental Results

[0092] A standard plasmid pAK-001 containing the internal reference gene alaA: the target gene bla: the target gene Kan in a ratio of 1:1:1 was successfully constructed. Its plasmid map is shown below. Figure 1 .

[0093] Example 2: Establishment of plasmid copy number qPCR methodology

[0094] 1. Primer and probe design

[0095] Primer and probe information are detailed in Tables 2 and 3.

[0096] Table 2 qPCR primer information

[0097]

[0098] Table 3 qPCR probe information

[0099]

[0100] 2. Preparation of standard curve samples and standards

[0101] The concentration of the standard is calculated using the following formula:

[0102] .

[0103] The pAK-001 standard quality particles were serially diluted tenfold, ranging from 1×10⁻⁶. 8 copies / μL ~1×10 3 copies / μL.

[0104] The selected concentration is 5×10 7 copies / μL, 5×10 5 Copies / μL were used as pAK-001 standard solution for verification.

[0105] 3. Amplification system (20 μL)

[0106] Sample amplification system: Probe qPCR Super PreMix 10 μL, PCR Forward Primer (10 μM) 0.5 μL, PCR Reverse Primer (10 μM) 0.5 μL, Probe (10 μM) 1 μL, template 2 μL, ddH2O 6 μL.

[0107] Negative control system: Probe qPCR Super PreMix 10 μL, PCR Forward Primer (10 μM) 0.5 μL, PCR Reverse Primer (10 μM) 0.5 μL, Probe (10 μM) 1 μL, ddH2O 8 μL.

[0108] 4. Amplification conditions

[0109] Step 1: 95℃ for 30 s; Step 2: 95℃ for 5 s; Step 3: 60℃ for 30 s; Step 2 to Step 3 are repeated 40 times, and fluorescence signals are received.

[0110] 5. Sample addition and testing

[0111] Each sample was made into three replicates (if the replicate SD ≤ 0.5, the average value of the three replicates was calculated; if the replicate SD > 0.5, the value with the largest deviation from the average value was removed, and the average value of the other two replicates was calculated).

[0112] 6. qPCR assay

[0113] A linear regression equation was constructed using log10 of the standard concentration as the x-axis and Ct as the y-axis, and the correlation coefficient was obtained. R 2 The experimental data are valid if the Ct value is ≥0.98, the slope is within the range of -3.1 to -3.8, the Ct value at the lowest point of the standard curve concentration is not higher than 39, and the Ct value of the negative control group, if present, is not lower than the Ct value at the lowest point of the standard curve concentration.

[0114] 7. Experimental Results

[0115] Experimental results are as follows Figure 2 As shown: The amplification efficiency of the real-time fluorescence PCR chemical method-TaqMan established in this experiment for detecting the internal reference gene alaA and the target genes bla and Kan of the standard was 83.30% to 110.17%.

[0116] As shown in Table 4, the copy numbers of the standard plasmid bla and Kan plasmid were both between 0.7 and 1.3, indicating that the standard contains the internal reference gene alaA: the test gene bla: the test gene Kan = 1:1:1. This can be used to detect the copy number of engineered plasmids containing ampicillin (or kanamycin) resistance genes in engineered bacterial strains.

[0117] Table 4. Results of Standard Granule Copy Number Detection

[0118]

[0119] Example 3: Methodological Validation (Taking plasmids containing ampicillin resistance as an example, the validation process for kanamycin resistance is similar and will not be shown separately here)

[0120] 1. Limit of Quantification Validation

[0121] Following the standard curve and operation steps established in Example 2, a 1×10⁻⁶ curve was selected. 3 The concentration of copies / μL is used as the proposed limit of quantitation for this method, and the accuracy and precision at this proposed limit of quantitation are determined.

[0122] As shown in Table 5: The two people respectively put 1×10 3 The pAK-001 standard solution was measured three times in six replicates, and the correlation coefficient R of the six validation results was obtained. 2 All values ​​were greater than 0.98, RSD was less than 30.0%, and recoveries were all between 50% and 150%, meeting the acceptable criteria. Therefore, 1×10⁻⁶ was used. 3 The limit of quantitation for this method is defined as copies / μL.

[0123] Table 5 Results of the Limit of Quantitation Validation Experiment

[0124]

[0125] 2. Range Verification

[0126] Select 1×10 8 copies / μL is used as the proposed upper limit of the range. Accuracy and precision are obtained by determining the values ​​at this proposed upper limit.

[0127] As shown in Table 6: Two researchers conducted a total of 6 experiments with R... 2 All values ​​are greater than 0.98, indicating the experimental data are valid. 1×10 8 copies / μL and 1×10 3 The RSDs of six determinations of the pAK-001 standard solution (copies / μL) were all less than 30.0%, and the recoveries were all between 50% and 150%, meeting the acceptable criteria. Therefore, the lower limit of the method range is 1×10⁻⁶. 3 copies / μL, with an upper limit of 1×10 8 copies / μL.

[0128] Table 6. Results of the range verification experiment

[0129]

[0130] 3. Linearity Validation

[0131] Following the standard curve and operation steps established in Example 2, a 1×10⁻⁶ curve was selected. 8 ~1×103 copies / μL is used as the standard curve range.

[0132] As shown in Table 7: The results of the six measurements indicate that when the standard concentration range is 1×10⁻⁶... 8 ~1×10 3 When copies / μL, this detection method R 2 All values ​​are greater than 0.98, which meets the acceptable standard, indicating that the method has good linearity.

[0133] Table 7 Results of Linearity Validation Experiments

[0134]

[0135] 4. Accuracy Verification

[0136] Following the standard curve established and operating procedures in Example 2, a concentration of 5 × 10⁻⁶ was selected. 6 copies / μL, 5×10 5 copies / μL, 5×10 4 The accuracy of the pAK-001 standard solution was verified by performing three assays using copies / μL of the solution. The recovery rates of the three assays were calculated.

[0137] As shown in Table 8, the test results of the three batches show that the recoveries of low, medium, and high concentrations of pAK-001 standard are all between 50% and 150%, and the correlation coefficient R is... 2 All values ​​are greater than 0.98, indicating that the method has high accuracy.

[0138] Table 8. Results of Accuracy Verification Experiment

[0139]

[0140] 5. Intermediate precision verification

[0141] The pET-mSEB / BL21 strain was streaked onto ampicillin plates and incubated upside down at 37°C for 14.5 h. Colonies from the plates were dissolved in ddH2O and analyzed using OD200. 600 The concentration was determined and diluted to 0.5 to prepare the test solution. Following the standard curve and operating procedures established in Example 2, two personnel collected the same sample at different times for testing.

[0142] As shown in Table 9, the relative standard deviation is within 15% (RSD≤30%), indicating that the method measured by the present invention has high intermediate precision.

[0143] Table 9 Results of intermediate precision verification experiments

[0144]

[0145] 6. Durability verification

[0146] The pET-mSEB / BL21 strain was streaked onto ampicillin plates and incubated upside down at 37°C for 14.5 h. Colonies from the plates were dissolved in ddH2O and analyzed using OD200. 600 The concentration was determined, and the solution was diluted to 0.5 to prepare the test solution.

[0147] Verification of pAK-001 standard (1×10) 8 copies / μL ~1×10 3 Did the incubation of six concentrations of standard (copies / μL) at 4°C affect the plasmid copy number determination results of the pET-mSEB / BL21 strain? The observation period was:

[0148] Condition 1: The standard is diluted before being added to the sample;

[0149] Condition 2: The standard sample was added after being placed at 4℃ for 1.5 hours;

[0150] Condition 3: The standard sample was added after being placed at 4℃ for 3 hours;

[0151] According to the standard curve and operating procedures established in Example 2, two people took the same sample to be tested at different times.

[0152] As shown in Table 10: the results of the three measurements indicate that R 2 All values ​​were greater than 0.98, indicating that the experimental data were valid. Under conditions 1, 2, and 3, the RSD of the test sample at the same concentration was ≤30.0%, which met the acceptable standard, indicating that the method has good robustness.

[0153] Table 10 Durability Verification Test Results

[0154]

[0155] 7. Specificity Verification

[0156] The standard pAK-001 contains both the target gene bla and the target gene Kan. To evaluate the specificity of the established method, the pET-28a plasmid containing Kan but not bla was used as the test sample to verify the specificity of the method.

[0157] As shown in Tables 11 and 12: Both the positive controls (bla and Kan genes) showed normal amplification. The Ct value of the Kan gene amplification in the pET-28a test sample was within the linear range of the standard curve, indicating normal Kan gene amplification. The Ct value of the bla gene in the pET-28a test sample was consistent with the negative result, being lower than the Ct value at the lowest concentration point of the standard curve, indicating no amplification. This demonstrates that this method can specifically detect the target gene bla, and Kan does not interfere with the detection results of the target gene bla. Correlation coefficient R 2 A value ≥0.98 meets the acceptable standard, indicating that the method has good specificity.

[0158] Table 11 Results of specific positive control amplification experiments

[0159]

[0160] Table 12 Results of Specific Sample Amplification Experiments

[0161]

[0162] Example 4: Determination of the copy number of the pET-mSEB / BL21 plasmid in recombinant Staphylococcus aureus vaccine (Escherichia coli) strain

[0163] 1. Experimental Methods

[0164] Streak pET-mSEB / BL21 onto ampicillin plates and incubate upside down at 37°C for 14.5 hours. Dissolve colonies from the plates in ddH2O and use OD200 to analyze the bacterial count. 600 The concentration was measured and diluted to 0.5 to serve as a template.

[0165] The copy number of the pGEX-MntC / XL plasmid of the recombinant Staphylococcus aureus vaccine (Escherichia coli) strain was determined 12 times according to the method and operation steps established in Example 2.

[0166] 2. Experimental Results

[0167] As shown in Table 13, the relative standard deviations of the 12 detection results were all controlled within 20%, and the average copy number of the target plasmid in this strain was 34.78, which is consistent with the characteristics of a high-copy-count plasmid. These results indicate that the detection method established in this study has good reproducibility and can be effectively used to determine the copy number of engineered plasmids containing ampicillin resistance genes in engineered bacterial strains, providing reliable detection technology support for subsequent related research or applications.

[0168] Table 13 Results of copy number determination of pET-mSEB / BL21 plasmid in recombinant Staphylococcus aureus vaccine (Escherichia coli)

[0169]

[0170] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Standard quality grains, characterized in that, The reference gene, the first gene to be tested, and the second gene to be tested contain the same copy number; The internal reference gene is a single-copy gene of engineered bacteria; The first and second genes to be tested are marker genes.

2. The standard quality grain as described in claim 1, characterized in that, The backbone is pGEX-6p-2, the internal reference gene is alaA, the first gene to be tested is bla, and the second gene to be tested is Kan.

3. The application of the standard plasmid as described in claim 1 or 2 in detecting the plasmid copy number of engineered bacteria.

4. The application as described in claim 3, characterized in that, The accuracy of the plasmid copy number is ensured based on the standard plasmid.

5. A method for detecting plasmid copy number in engineered bacteria based on qPCR, characterized in that, include: S1: Use standard quality particles to verify the amplification efficiency of the internal reference gene primer and probe set, the first test gene primer and probe set, and the second test gene primer and probe set. S2: Using the internal reference gene primer and probe set, the internal reference gene of the engineered bacteria to be tested is amplified to obtain the Ct value of the internal reference gene; S3: Using the first test gene primer and probe set as a template, the first test gene on the plasmid of the engineered bacteria is amplified to obtain the Ct value of the first test gene; S4: Using the second test gene primer and probe set as a template, the second test gene on the plasmid of the engineered bacteria is amplified to obtain the Ct value of the second test gene; S5: Calculate the plasmid copy number based on at least one of the following: a) The Ct value of the internal reference gene and the Ct value of the first gene to be tested; b) The Ct value of the internal reference gene and the Ct value of the second gene to be tested; The standard quality grain contains the internal reference gene, the first gene to be tested, and the second gene to be tested, all in equal copy numbers.

6. The method as described in claim 5, characterized in that, After S2 and before S3, it also includes: Verify the accuracy of plasmid copy numbers obtained based on the internal reference gene primer and probe set, the first test gene primer and probe set, and the second test gene primer and probe set using any of the following methods: i) Determine the copy number of the plasmid; ii) Engineered bacteria containing plasmids with a defined copy number.

7. The method as described in claim 5, characterized in that, In S1, the amplification efficiencies of both the internal reference gene primer and probe set and the first target gene primer and probe set are between 83.30% and 110.17%. The amplification efficiencies of both the internal reference gene primer and probe set and the second test gene primer and probe set ranged from 83.30% to 110.17%.

8. The method as described in claim 5, characterized in that, The internal reference gene is a single-copy gene of engineered bacteria; The first and second genes to be tested are marker genes.

9. The method as described in claim 8, characterized in that, The internal reference gene is alaA, the first gene to be tested is bla, and the second gene to be tested is Kan.

10. The method as described in claim 1, characterized in that, The backbone of the standard quality grain is pGEX-6p-2.

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