Primer pair, detection reagent and detection method for detecting residual DNA of Streptomyces roseosporus

Abstract

The present invention relates to the field of biological detection, and in particular to a primer pair for detecting residual DNA of Streptomyces roseosporus, a detection reagent and a kit comprising the primer pair, and a detection method for residual DNA of Streptomyces roseosporus using the primer pair, wherein the primer pair specifically binds to the sequence shown in SEQ ID NO: 1. The primer pair in the present invention can specifically bind to the residual DNA of Streptomyces roseosporus, and can distinguish other eukaryotic cells without interfering with other DNA. At the same time, it has extremely high precision, can still maintain linearity requirements within a wide concentration range, and has good repeatability. In addition, fragmentation of the Streptomyces roseosporus DNA reference does not affect qPCR detection, and the detection method also has the advantages of being simple and quick to operate.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and in particular to primer pairs, detection reagents, and detection methods for detecting residual DNA in Streptomyces roseosus. Background Art

[0002] Daptomycin is a novel cyclic lipopeptide antibiotic that primarily works by disrupting the transport of amino acids across the cell membrane, thereby inhibiting the biosynthesis of peptidoglycan in the bacterial cell wall and altering the properties of the cytoplasmic membrane. It can damage bacterial cell membrane function in multiple ways and rapidly kill Gram-positive bacteria. Due to its unique structure and special mechanism of action, it is considered the last line of defense against severe infections caused by multidrug-resistant Gram-positive bacteria. Daptomycin exhibits excellent antibacterial activity against antibiotic-resistant bacteria.

[0003] Microbial fermentation is a simple, rapid, high-yield, and low-contamination method, making it the mainstream method used for large-scale daptomycin production. Daptomycin is primarily produced by *Streptomyces roseosus* (…). Streptomyces roseosporus Daptomycin is obtained through a semi-synthetic method after fermentation. However, genomic DNA of *Streptomyces roseospora* may remain after fermentation, posing potential risks such as infectivity, carcinogenicity, immunogenicity, and mutagenicity. Therefore, the detection of residual *Streptomyces roseospora* genomic DNA in daptomycin is crucial.

[0004] Real-time quantitative polymerase chain reaction (qPCR) involves adding a fluorescent group to the PCR reaction system, combining traditional endpoint detection PCR with fluorescence detection technology. Based on the principle that fluorescence is directly related to the amount of double-stranded DNA in the amplification reaction, the entire PCR process is monitored in real time using the accumulation of fluorescence signals, enabling the quantification of target nucleic acid fragments. This method can also be used to quantify unknown templates using a standard curve. Due to its advantages of high specificity, high sensitivity, good accuracy, and ease of operation, this method has become the technological trend for detecting host residual DNA in biological products. It has been recognized as a mainstream technology by relevant regulatory agencies in various countries and is a recommended method in the latest pharmacopoeias of Europe and the United States.

[0005] The WHO, FDA, and my country's drug regulatory agencies all have limits on DNA residue in biological products. For non-oral biological products, the WHO and my country limit residual DNA to no more than 10 ng / dose, while the FDA limits it to 100 pg / dose. Summary of the Invention

[0006] The purpose of this invention is to provide a primer pair, detection reagent, and method for detecting residual DNA of Streptomyces roseosus in biological products.

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

[0008] In a first aspect, the present invention provides a primer pair for detecting residual DNA in *Streptomyces roseosus*.

[0009] The primer pair includes a forward primer and a reverse primer, wherein:

[0010] The forward primer is shown in SEQ ID NO:2;

[0011] The reverse primer is shown in SEQ ID NO:3.

[0012] In a second aspect, the present invention provides a detection reagent for detecting residual DNA in Streptomyces roseosus, the detection reagent comprising the primer pair as described in the first aspect.

[0013] In a specific implementation, the forward primer is shown in SEQ ID NO:2, and the reverse primer is shown in SEQ ID NO:3.

[0014] In a specific implementation, the detection reagent also includes a probe.

[0015] Preferably, the probe tip of the detection reagent is labeled with a fluorescent reporter group FAM and a quencher group TAMRA.

[0016] Preferably, the probe in the detection reagent is shown in SEQ ID NO:4.

[0017] Preferably, the sensitivity of the detection reagent is 0.03 pg / μL.

[0018] In a third aspect, the present invention also provides a detection kit for residual DNA of Streptomyces roseosus, comprising the detection reagents described above.

[0019] Preferably, the detection kit further includes: qPCR reaction buffer, DNA dilution solution, DNA quantification reference, and sample extraction and purification solution.

[0020] In a fourth aspect, the present invention also provides a method for detecting residual DNA in Streptomyces roseosus.

[0021] The method includes: performing qPCR on the sample to be tested using the primer pairs, detection reagents, or detection kits described above, and detecting the qPCR amplification products.

[0022] As a preferred method, the reaction procedure for qPCR is as follows: 95℃ pre-denaturation for 10 min; 95℃ for 15 s; 60℃ for 1 min, 40 cycles.

[0023] In a fifth aspect, the present invention also provides the use of the primer pairs, detection reagents, or detection kits described above, primarily for detecting the presence of Streptomyces roseosus residual DNA in the test subject.

[0024] Preferably, the present invention is for detecting the presence of residual DNA of Streptomyces roseosporus in daptomycin.

[0025] Therefore, the present invention has the following beneficial effects:

[0026] (1) The primer pair in this invention can specifically bind to residual DNA of Streptomyces roseosus and can distinguish other eukaryotic cells without interfering with other DNA;

[0027] (2) It also has extremely high precision, can maintain linearity over a wide concentration range, and has good repeatability;

[0028] (3) In addition, the fragmentation of the Streptomyces roseosporus DNA reference did not affect the qPCR detection;

[0029] (4) The detection method of the present invention also has the advantages of being simple and quick to operate. Attached Figure Description

[0030] Figure 1 The standard curve is used as a reference.

[0031] Figure 2 The amplification curve is for reference.

[0032] Figure 3 This is a diagram showing the specific results of utilizing the genomic DNA of Pichia pastoris, Escherichia coli, CHO cells, Vero cells, and zoonotic actinomycetes.

[0033] Figure 4 The results show the electrophoresis of DNA fragments with different degrees of fragmentation; lane 1 is the DNA marker, with values ​​of 2000, 1000, 750, 500, 250, and 100 bp from top to bottom; lanes 2, 3, and 4 are reference samples of Streptomyces roseospora DNA, sonicated for 0, 1, and 10 min, respectively.

[0034] Figure 5 This is the result of qPCR detection of a series of DNA fragments with different degrees of fragmentation after ultrasonication for 0 min, 1 min, and 10 min respectively. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0036] The primer pairs of the present invention

[0037] The term "primer" as used in this invention has the meaning conventionally understood by those skilled in the art. The *Streptomyces roseospora* residual DNA-specific primers of this invention are not designed for the exogenous gene itself or the viral vector itself, but rather for the sequence shown in SEQ ID NO:1 of the *Streptomyces roseospora* cell genomic DNA. In other words, the primers of this invention can specifically bind to the sequence shown in SEQ ID NO:1 of the *Streptomyces roseospora* cell genomic DNA.

[0038] The specific sequence of the genomic DNA of Streptomyces roseosus cells, shown in SEQ ID NO:1, is as follows:

[0039]

[0040] In view of the teachings of this invention and common knowledge in the art, those skilled in the art should understand that various primer pairs can be designed for the sequence shown in SEQ ID NO:1. Therefore, the primer pairs of this invention are not limited to the specific primer pairs shown in the embodiments.

[0041] In a specific embodiment, the forward primer in the primer pair of the present invention is as shown in SEQ ID NO:2.

[0042] The sequence shown in SEQ ID NO:2 is: CGGCGATGTTCCAGGTTAGTT.

[0043] In a specific embodiment, the reverse primer has the sequence shown in SEQ ID NO: 3.

[0044] The sequence shown in SEQ ID NO: 3 is specifically: TGCTGGATCGTAGGCACTACTTC.

[0045] The probe of the present invention

[0046] The term "probe" described herein has the meaning commonly understood by those skilled in the art, ie, a short single-stranded DNA or RNA fragment used to detect a nucleic acid sequence complementary thereto.

[0047] Based on the teachings of this invention and common knowledge in the art, those skilled in the art should understand that, knowing the primer pairs, they can independently design probes based on the template sequence between the binding sites of the forward and reverse primers, and test the technical effect of the probes with the primer pairs. In specific embodiments, those skilled in the art can design probes as needed; the probes can be in a liquid phase or immobilized on a solid phase; they can bind before or after amplification. Therefore, the probes of this invention are not limited to the probes specifically disclosed in the embodiments. The primer pairs of this invention are also not limited to use in conjunction with the probes specifically disclosed in the embodiments.

[0048] In a specific embodiment, the probe of the present invention is as shown in SEQ ID NO:4.

[0049] The sequence shown in SEQ ID NO:4 is specifically: FAM-CACGAACGGCACACAGAAACCGG-TAMRA.

[0050] The detection reagent of the present invention

[0051] The present invention also provides a detection reagent for residual DNA of Streptomyces roseosporus, which comprises the primer pair and probe of the present invention and other components required for implementing PCR, such as qPCR reaction buffer, DNA diluent, DNA quantitative reference, and sample extraction and purification solution.

[0052] In a specific implementation:

[0053] The primer pair comprises a forward primer as shown in SEQ ID NO: 2 and a reverse primer as shown in SEQ ID NO: 3.

[0054] The probe has the sequence shown in SEQ ID NO: 4.

[0055] In a specific embodiment, the detection sensitivity of the detection reagent of the present invention reaches 0.03 pg / μL.

[0056] Based on the primer pair or detection reagent of the present invention, the present invention provides a detection kit for residual DNA of Streptomyces roseosporus, which comprises a container and the primer pair of the present invention located in the container.

[0057] In a specific embodiment, the kit of the present invention further comprises: qPCR reaction buffer, DNA diluent, DNA quantitative reference substance and sample extraction and purification solution.

[0058] Based on the primer pair or detection reagent of the present invention, the present invention further provides a method for detecting residual DNA of Streptomyces roseosporus, which comprises using the primer pair or detection reagent or detection kit as described above to perform qPCR on the sample to be tested, and detecting the qPCR amplification product.

[0059] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0060]

Experimental Materials

[0061] 1.1 Sample pretreatment reagents:

[0062] Proteinase K, Proteinase K buffer, magnetic beads, binding buffer, glycogen, yeast tRNA, wash buffer A, wash buffer B, eluent.

[0063] 1.2 qPCR detection system:

[0064] qPCR reaction buffer, DNA dilution solution, primer pairs of the present invention, probes of the present invention, DNA quantification reference, sample extraction and purification solution, wherein the target sequence, primer pairs and probes of the present invention are specifically shown in Table 1 below.

[0065] Table 1

[0066]

[0067]

Experimental methods

[0068] 2.1 Sample processing and extraction

[0069] Accurately weigh 10 mg of the dry powder sample and place it in a 1.5 mL DNase-free sterile centrifuge tube.

[0070] 2) Take 100 μL for the determination of DNA residue in the sample and 100 μL for spike recovery (300 pg and 30 pg of Streptomyces roseosporus genomic DNA).

[0071] 3) Add 10 μL of 5 M NaCl, 10 μL of proteinase K, and 100 μL of proteinase K buffer to the sample, mix well, and digest in a 55°C metal bath for 1 h.

[0072] 4) Add 200 μL of working binding buffer, 9 μL of glycogen, and 0.2 μL of yeast tRNA to the digested sample and mix well.

[0073] 5) Add 200 μL of isopropanol and 15 μL of magnetic beads, vortex for 5 minutes to mix, centrifuge quickly for 10 seconds, and place on a magnetic stand. Once the solution is clear and the beads are completely separated, slowly open the tube cap and carefully aspirate the supernatant.

[0074] 6) Add 700 μL of Wash Buffer A, vortex to mix, centrifuge quickly for 10 seconds, and place on a magnetic stand. Once the solution is clear and the beads are completely separated, aspirate the supernatant.

[0075] 7) Add 700 μL of Wash Buffer B, vortex to mix, centrifuge quickly for 10 seconds, and place on a magnetic rack. Once the solution is clear and the beads are completely separated, aspirate the supernatant.

[0076] 8) Centrifuge again for 10 seconds, place on a magnetic rack, and remove any remaining liquid after the magnetic beads have completely separated.

[0077] 9) Open the tube cap and dry at room temperature for 5-10 seconds to remove any residual ethanol. Observe the beads carefully during the drying process to avoid excessive drying, as this may prevent them from completely dissolving during elution.

[0078] 10) Add 50 μL of elution buffer, gently shake to mix the magnetic beads and elution buffer, incubate in a 70 ℃ water bath for 7 min, shaking once every 2 min during the water bath, and shaking to mix 2-3 times.

[0079] 11) Centrifuge quickly for 10 seconds, place on a magnetic rack, and after the magnetic beads separate, transfer the purified supernatant of the sample to a new centrifuge tube.

[0080] 2.2 Detection system

[0081] 1) Preparation of reference materials

[0082] The reference sample was extracted genomic DNA from Streptomyces roseosus, with a concentration of 30 ng / μL.

[0083] 2) Sample preparation for the calibration curve

[0084] The reference sample was serially diluted 10-fold using DNA dilution buffer to prepare standard solutions of 3000 pg / μL, 300 pg / μL, 30 pg / μL, 3 pg / μL, 0.3 pg / μL, and 0.03 pg / μL.

[0085] 3) Reaction system

[0086] The detection system consisted of 30 μL of: 20 μL qPCR MIX (containing 17 μL qPCR Reaction Buffer; 0.06 μL primers and probes each; and 2.82 μL DNA dilution) + 10 μL template. See Table 2 below for sample loading instructions for each reaction well.

[0087] Table 2

[0088]

[0089] 4) Reaction procedure

[0090] Pre-denaturation at 95℃ for 10 min; 95℃ for 15 s; 60℃ for 1 min, 40 cycles.

[0091] 5) Result Determination

[0092] a) Correlation coefficient R of the standard curve equation 2 It should be greater than 0.990, with a slope between -3.1 and 3.8 (i.e., amplification efficiency between 83.3% and 110%).

[0093] b) The Ct value was not detected or was ≥35 in the template-free control (NTC).

[0094]

Experimental results

[0095] 4.1 Linear Range (Genomic DNA)

[0096] The DNA standard curve was 300 pg / μL, 30 pg / μL, 3 pg / μL, 300 fg / μL, and 30 fg / μL, and the CT value test results are shown in Table 3 below.

[0097] Table 3

[0098]

[0099] Figure 1 is the standard curve of the reference product. Figure 1 It can be seen that when the concentration of the reference substance is 300pg / μL~300fg / μL, the correlation coefficient R of the standard curve is 2 =99.8%, amplification efficiency E=97.9%, and the standard curve had good linearity, which met the standard requirements.

[0100] Figure 2 The amplification curve for the reference sample shows a clear exponential growth phase and is still detectable at 0.03 pg / μL, indicating that the sensitivity can reach 0.03 pg / μL.

[0101] 4.2 Specificity

[0102] 30pg / μL of Pichia pastoris, Escherichia coli, CHO cells, Vero cells and Actinomycetes genomic DNA were detected respectively. The test results are as follows: Figure 3 As shown in the curves, it can be seen that the genomic DNA of Pichia pastoris, Escherichia coli, CHO cells, Vero cells and Actinomycetes planaris has no effect on the detection of the primer pair of the present invention (SEQ ID NO: 2 and SEQ ID NO: 3), and the detection Ct values ​​are all Undet, indicating that the primer set has excellent specificity.

[0103] 4.3 Precision

[0104] Repeated tests were performed on Streptomyces roseosus DNA at a concentration of 3 pg / μL. The results are shown in Table 4 below. The results of the reference sample show that its CV = 1.724%, which meets the standard requirements.

[0105] Table 4

[0106]

[0107] 4.4 Recovery rate

[0108] The recoveries were tested in 300 pg and 30 pg samples, and the results are shown in Table 5 below. The results show that the recoveries in 300 pg and 30 pg samples were 100-130%, with CV < 15.

[0109] Table 5

[0110]

[0111] 4.5 Fragmentation Experiment

[0112] The *Streptomyces roseosus* DNA reference sample was sonicated for 0 min, 1 min, and 10 min, and the degree of fragmentation was detected by electrophoresis. The DNA from different sonication times (300 pg / μL, 30 pg / μL, 3 pg / μL, 0.3 pg / μL, and 0.03 pg / μL) was serially diluted and amplified by qPCR. The results are as follows: Figure 4 As shown in Figure 5, fragmentation of the *Streptomyces roseosus* DNA reference does not affect qPCR detection.

[0113] 4.6 Sample Testing

[0114] Based on the sample pretreatment and qPCR detection methods established above, trace amounts of DNA were extracted from three samples from different batches and subjected to qPCR reactions. The experimental results are shown in Table 6 below.

[0115] As can be seen from the data in the above table, no genomic DNA of Streptomyces roseosporus was detected in the three daptomycin samples of different batches. Sequence Listing <110> Huzhou Shenke Biotechnology Co., Ltd. <120> Primer pairs, reagents, and detection methods for detecting residual DNA in Streptomyces roseospora. <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1294 <212> DNA <213> Artificial sequence <400> 1 ggatccggca accgcgggga cggccggcgg ccaggcgttc cggcccgtcg ccggtggtcg 60 gcggccctac cccgcgggcg tggacagcat ttgttttgac ccagctccgt gaggtaggta 120 cgctcaagcc ttgtgcctgg ggtgtgcctg ggctcgtgtg cgtgtcctca accgcagcgc 180 gagtccgtca gtggccaccg caatctgcgc cccttctgcc ccagggcggg agtccgcagt 240 attcgacaca cccgaccgcg tgggtcggcg atgttccagg ttagtttcac gaacggcaca 300 cagaaaccgg agaagtagtg cctacgatcc agcagctggt ccggaagggc cggcaggaca 360 aggtcgagaa gaacaagacg cccgcgctcg agggttcgcc ccagcgtcgc ggtgtctgca 420 cgcgtgtgtt cacgaccacc ccgaagaagc cgaactcggc gctccgtaag gtcgcgcgtg 480 tgcgtctgac ctccggcatc gaggtcacgg cctacatccc gggtgaggga cacaacctgc 540 aggagcactc catcgtgctc gtgcgtggtg gccgtgtgaa ggacctgccg ggtgttcgtt 600 acaagatcat ccgcggttcg ctcgacaccc agggtgtcaa gaaccgcaag caggcccgca 660 gccgctacgg cgccaagaag gagaagtaag aatgcctcgt aagggccccg ccccgaagcg 720 cccggtcatc atcgacccgg tctacagctc tcctcttgtc acctcgctga tcaacaagat 780 cctcctcgac ggcaagcgtt ccaccgccga gcggatcgtg tacggcgcca tggaaggcct 840 ccgcgagaag accggcaacg acccggtcat cacgctgaag cgcgcgcttg agaacgtcaa 900 gccctcgctc gaggtcaagt cccgccgtgt cggtggcgcc acctaccagg tgccgatcga 960 ggtcaagccc ggtcgcgcct ccaccctcgc tctgcgctgg ctcgtcggtt actcccgcgc 1020 ccgccgcgag aagaccatga ccgagcgcct catgaacgaa ctgctcgacg cctccaacgg 1080 cctcggtgct tcggtcaaga agcgtgagga cacccacaag atggccgagt ccaacaaggc 1140 cttcgcgcac taccgctggt agtcgctacc cccatcgaga ccgagagaag actgagcctt 1200 atggccacca cttcgcttga cctggccaag gtccgcaaca ttgggatcat ggcccacatc 1260 gacgcgggca agacgaccac cactgagcgg atcc 1294 <210> 2 <211> 21 <212> DNA <213> Artificial sequence <400> 2 cggcgatgtt ccaggttagt t 21 <210> 3 <211> 23 <212> DNA <213> Artificial sequence <400> 3 tgctggatcg taggcactac ttc 23 <210> 4 <211> 23 <212> DNA <213> Artificial sequence <400> 4 cacgaacggc acacagaaac cgg 23

Claims

1. Use of a primer pair and a probe in the preparation of a reagent for detecting residual DNA of Streptomyces roseosporus in daptomycin, characterized in that: The primer pair comprises a forward primer and a reverse primer, wherein: The forward primer is shown in SEQ ID NO: 2; The reverse primer is shown in SEQ ID NO: 3; The probe is shown in SEQ ID NO:

4.

2. Use of the primer pair and probe according to claim 1 in the preparation of a kit for detecting residual DNA of Streptomyces roseosporus in daptomycin.

3. The use according to claim 2, characterized in that The kit also includes: qPCR reaction buffer, DNA diluent, DNA quantitative reference and sample extraction and purification solution.

4. A method for detecting residual DNA of Streptomyces roseosporus in daptomycin, characterized in that: The method comprises: performing qPCR on a sample to be tested using the reagent according to claim 1 or the kit according to any one of claims 2 to 3, and detecting a qPCR amplification product.

5. Use of the reagent according to claim 1 or the kit according to any one of claims 2 to 3 for detecting whether residual DNA of Streptomyces roseosporus is present in daptomycin.

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