A positive quality control product for detecting group B streptococci and its preparation method

Abstract

The present invention relates to the field of biotechnology, and in particular to a positive quality control product for detecting group B streptococci and a preparation method thereof. The quality control product comprises an inactivated GBS strain, an internal standard plasmid, and a diluent; the inactivated GBS strain comprises at least one of types Ia, Ib, III, and V; and the diluent is composed of P300, P950, silicone, EDTA·2Na, and HEPES buffer. Experimental results show that the quality control product provided by the present invention has good stability, can be stably stored for more than 14 days at 37°C for thermal acceleration, and can be stably stored for at least 18 months at 2-8°C, with significantly better stability than existing products. The quality control product is consistent with actual GBS clinical swab samples and can be detected by marketed manufacturers as a standard quality control product. It can realistically simulate the in vitro nucleic acid detection process of GBS and has strong versatility. The product has the advantages of low cost, high stability, and easy storage and transportation, making it convenient for large-scale industrial production and has important value and application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a positive quality control product for detecting group B streptococci and a preparation method thereof. Background Art

[0002] Group B Streptococci (GBS) are facultative, anaerobic, Gram-positive streptococci. They are asymptomatic bacteria common to the intestinal and urogenital tracts and are opportunistic pathogens. They normally reside in the vagina and rectum, with a carrier rate exceeding 30% in healthy women. GBS infection in pregnant women can be transmitted to newborns through the birth canal, causing neonatal sepsis, meningitis, pneumonia, and other conditions. It is currently one of the leading pathogens causing neonatal infections.

[0003] In 1996, the U.S. Centers for Disease Control and Prevention (CDC) and other agencies jointly developed the "Guidelines for Screening and Prevention of Perinatal Group B Streptococcal Infections." The CDC revised these guidelines twice, in 2002 and 2010, ultimately deciding to adopt a universal screening program for the prevention and treatment of GBS in pregnant women. These guidelines state that at the time of delivery, pregnant women with no or unknown culture results and any of the following risk factors (gestational age at delivery <37 weeks, rupture of membranes ≥18 hours, antepartum temperature ≥38°C) should be promptly treated. For pregnant women without any risk factors, direct vaginal and rectal sampling for nucleic acid amplification can be performed to rapidly detect GBS positive or negative results.

[0004] Nucleic acid testing primarily monitors GBS infection in patients by specifically amplifying GBS target DNA in genital and rectal swabs of pregnant women. Group B Streptococcus nucleic acid testing quality control products are used for quality control of GBS nucleic acid testing. However, current GBS quality control products used in clinical laboratories are primarily ancillary products to marketed nucleic acid testing products. These products suffer from numerous instabilities, including poor stability, large batch-to-batch variability, and inconsistent target values calibrated across different units. Furthermore, the active ingredients of positive quality control products in various manufacturers' kits are plasmids containing target sequences or recombinant strains constructed using these target plasmids. The target sequences amplified by each manufacturer vary, and these products can only be amplified and detected by the amplification reagents in their own kits and cannot be used for quality control of kits from other manufacturers. This significantly hinders the uniformity and standardization of GBS nucleic acid testing quality control. Furthermore, there is currently no independent manufacturer of quality control products in China, and these products must be stored at -20°C, making them inconvenient to use. Furthermore, repeated freeze-thaw cycles significantly affect test results. Therefore, there is an urgent need for a versatile, stable positive quality control product for Group B Streptococcus nucleic acid testing. Summary of the Invention

[0005] In view of this, the present invention provides a positive quality control product for detecting group B streptococci and a preparation method thereof. The positive quality control product has good stability, versatility and consistency.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A positive quality control product for detecting group B streptococci, including an inactivated GBS strain, an internal standard plasmid, and a diluent;

[0008] The inactivated GBS strain includes at least one of types Ia, Ib, III, and V;

[0009] The diluent consists of P300, P950, silicone, EDTA·2Na and HEPES buffer.

[0010] In a specific embodiment, the present invention investigates the accelerated stability and long-term stability of positive quality control products prepared with different diluents. The results show that the positive quality control products prepared with the diluent of the present invention can effectively prevent the degradation of the quality control product target and internal standard substance, provide a stable environment for the quality control product, and extend the shelf life of the quality control product. The quality control product for group B streptococcus nucleic acid detection prepared using the quality control product preservation solution is not easily degraded and can be stably stored.

[0011] In the positive quality control product provided by the present invention, the percentage of each component in the diluent is:

[0012] 0.1-0.2 vol% P300, 0.15-0.3 vol% P950, 0.01-0.02 vol% silicone defoamer and 0.035-0.07 wt% EDTA·2Na.

[0013] In some specific embodiments, the percentage of each component in the diluent is:

[0014] 0.1 vol% P300, 0.15 vol% P950, 0.01 vol% silicone, and 0.035 wt% EDTA·2Na;

[0015] Or, 0.2 vol% P300, 0.3 vol% P950, 0.02 vol% silicone and 0.07 wt% EDTA·2Na.

[0016] In the present invention, the internal standard plasmid is a plasmid containing human β-globin gene.

[0017] Furthermore, the human β-globin gene includes at least one gene with sequence ID MK476491.1, MK476483.1, and MK476464.1.

[0018] In the present invention, the concentration of the inactivated GBS strain and the internal standard plasmid of the human β-globin gene can be selected according to clinical needs, and is generally a low to medium concentration in clinical practice. In the present invention, the concentration of the inactivated GBS strain in the positive quality control product is preferably 1×10 4 ~1×10 5 CFU / mL; the concentration of the internal standard plasmid with human β-globin gene in the positive quality control product is 1×10 4 ~1×10 5 copies / mL.

[0019] The present invention also provides a method for preparing the positive quality control product, comprising the following steps:

[0020] Activate, subculture, and expand the GBS strain; obtain a GBS strain suspension by centrifugation, resuspending, and inactivating;

[0021] The GBS strain suspension and the internal standard plasmid solution containing the human β-globin gene were mixed, and P300, P950, silicone defoamer and EDTA·2Na were added to obtain a positive quality control product.

[0022] The present invention also provides the use of the positive quality control product in preparing a nucleic acid detection kit for detecting group B streptococci.

[0023] In a specific embodiment, the present invention has experimentally found that the positive quality control products of various manufacturers on the market can only be amplified and detected by their respective matching test kits, and cannot be amplified and detected by test kits of other manufacturers; the positive quality control product for GBS nucleic acid detection prepared by the present invention can be amplified by test kits of all manufacturers, indicating that the quality control product of the present invention can be used as a universal positive quality control material and can be widely used for quality control in GBS nucleic acid detection.

[0024] The quality control product provided by this invention exhibits excellent stability, remaining stable for over 14 days at 37°C and for at least 18 months at 2-8°C, significantly surpassing existing products in terms of stability. This quality control product is consistent with actual GBS clinical swab samples, exhibits strong versatility, and can realistically simulate the in vitro GBS nucleic acid detection process, thereby enabling comprehensive monitoring of GBS nucleic acid testing. Furthermore, the product offers advantages such as low cost, high stability, and ease of storage and transportation, making it suitable for large-scale industrial production and possessing significant value and application prospects. DETAILED DESCRIPTION

[0025] The present invention provides a positive quality control product for detecting group B streptococci and a method for preparing the same. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0026] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0027] The present invention will be further described below in conjunction with the embodiments:

[0028] Example 1 Preparation of diluent and quality control products of the present invention

[0029] The diluent composition is: 0.1 vol% P300, 0.15 vol% P950, 0.01 vol% silicone defoamer, 0.035 wt% EDTA·2Na and HEPES buffer.

[0030] Positive quality control: GBS strain containing the target nucleic acid sequence - concentration 1×10 4 CFU / mL, human β-globin gene plasmid - concentration 1×10 4 copies / mL, 0.1 vol% P300, 0.15 vol% P950, 0.01 vol% silicone antifoaming agent, 0.035 wt% EDTA·2Na and HEPES buffer.

[0031] The preparation method of positive quality control includes the following steps:

[0032] (1) Preparation of human β-globin gene plasmid: Based on the human β-globin gene nucleic acid sequence in the NCBI database, conserved primer sequences were designed to synthesize the target gene. PCR amplification was performed using the nucleic acid containing the target gene sequence as a template. The primer sequences are shown in Table 2. The PCR product was subjected to agarose gel electrophoresis, the band containing the target gene fragment was cut from the gel, and the target gene was recovered using an agarose gel recovery kit. ② Construction and verification of the plasmid vector containing the target gene: The target gene fragment and the vector were double-digested with restriction endonucleases, the digested products were detected by agarose gel electrophoresis, and the gel was cut and recovered. The digested products were ligated to obtain ligation products, which were then transformed into competent cells, spread on LB culture plates, and inverted at 37°C to select single white positive colonies. DNA of the positive colonies was extracted and verified by PCR. Colonies verified by PCR as positive for the target gene were sequenced for verification. ③ Preparation of the target gene plasmid solution: Inoculate a single positive colony confirmed by sequencing into LB liquid medium. After incubation, centrifuge, discard the supernatant, and retain the precipitate. Add ultrasonic buffer (50 mmol / L Tris-HCl, pH 8.0; 2 mmol / L EDTA; 20% glycerol; 100 mmol / L NaCl; 0.1% Triton X-100) and sonicate in an ice bath. Ultrasonication conditions: 350W, 5s pause, 5s sonication, 30 cycles. Then, centrifuge the sonicated product at 6000 rpm for 10 minutes, and collect the supernatant, which is the plasmid solution. ④ Preparation of the quality control: Add RNase I to the plasmid solution to digest the RNA in the solution. Dilute to a specific concentration to obtain the human β-globin gene plasmid.

[0033] Table 1 Human β-globin gene sequence information

[0034]

[0035]

[0036] (2) Preparation of positive quality control products: ① Preparation of GBS strains containing target nucleic acid sequences: GBS Ia, Ib, III, and V strains were purchased from the ATCC Culture Collection in the United States. The corresponding strain numbers are shown in Table 3. The lyophilized powder of each strain was added to 1 mL of physiological saline for activation. After oscillation on a rapid mixer for 1-2 minutes, 20 μL of the activated bacterial solution was spread on a group B Streptococcus chromogenic plate (Antu Biological) for streak culture. A total of 3 blood agar plates were spread. After culturing in a 37°C constant temperature incubator for 24 hours, the strains were identified as GBS according to the instructions of the group B Streptococcus chromogenic plate. The colonies on the plate were picked and inoculated into 30 mL of LB liquid culture medium. After culturing in a 37°C constant temperature incubator for 36 hours, the strains were placed on a rapid mixer and cultured. The GBS culture medium was centrifuged at 6000 rpm for 10 minutes, the supernatant was collected, and the suspension was resuspended in sterile physiological saline to obtain the GBS strain suspension. ② Inactivation and verification of GBS strain suspension containing target nucleic acid sequence: Place the GBS strain suspension in a 60℃ water bath and heat for 1 hour to inactivate it. Take 0.1mL of the inactivated GBS strain suspension and add it to the group B streptococcus color plate. Spread 3 plates in total and place them in a 37℃ incubator for 24 hours. After that, observe and confirm that no colonies grow, which proves that the inactivation effect is qualified. ③ Preparation of quality control product: Add the plasmid solution containing human β-globin gene obtained in step (1) (concentration 1×10 4 copies / mL) and the inactivated GBS strain suspension containing the target nucleic acid sequence was diluted to a certain concentration (1×10 4 CFU / mL), add it to P300, P950, silicone defoamer and EDTA·2Na to obtain.

[0037] Table 2 Origins of the four common GBS serotypes

[0038] GBS strain serotype ATCC number Type Ia ATCC 31574 Type Ib ATCC 12401 Type III ATCC 31577 V-type ATCC BAA-611

[0039] Example 2 Preparation of diluent and quality control products of the present invention

[0040] The diluent composition is: 0.2 vol% P300, 0.3 vol% P950, 0.02 vol% silicone defoamer, 0.07 wt% EDTA·2Na and HEPES buffer.

[0041] Positive quality control: GBS strain containing the target nucleic acid sequence - concentration 1×10 5 CFU / mL, human β-globin gene plasmid - concentration 1×10 5 copies / mL, 0.2 vol% P300, 0.3 vol% P950, 0.02 vol% silicone antifoaming agent, 0.07 wt% EDTA·2Na and HEPES buffer.

[0042] The preparation method of the positive control product is the same as that in Example 1.

[0043] Test Example 1 Comparison of the stability of quality control products with different dilution compositions

[0044] This experiment investigated the stability of 8 different dilution control products in Comparative Examples 1 to 7 and Example 1. The specific compositions are shown in Table 3:

[0045] Table 3 Formulas of different quality control dilutions

[0046]

[0047] 2. Study on the stability of quality control product diluent

[0048] Quality control products were prepared according to the diluent formula in Table 3 to obtain quality control products of Comparative Examples 1 to 7, wherein the difference between the quality control products of Comparative Examples 1 to 7 and Example 1 was only the different diluent formulas, and other components and conditions were the same.

[0049] The above quality control products were placed at 37°C for 3, 7, and 14 days to assess the thermal stability of the prepared GBS quality control products. The thermal stability was compared with the Ct value on day 0 to screen out the formula with the smallest Ct difference (the smallest change in plasmid concentration). The assessment data are shown in Table 4 below:

[0050] Table 4 Accelerated validation of quality control products prepared with different dilutions at 37°C

[0051]

[0052] The results showed that after 14 days of thermal acceleration, the target and internal standard Ct changes of the low-concentration quality control prepared with the diluent provided in Example 1 were significantly lower than those of the solutions in Comparative Examples 1 to 7 compared to day 0. This indicates that the quality control prepared with the diluent provided by the present invention has the highest stability compared to other diluents.

[0053] Test Example 2: Comparison of the stability of quality control products with different dilution compositions

[0054] This experiment investigated the stability of 8 different dilution quality control products in Comparative Examples 1 to 7 and Example 2. The specific compositions are shown in Table 3 above.

[0055] The stability assessment plan is the same as that of Test Example 1. The assessment data are shown in Table 5 below:

[0056] Table 5 Accelerated validation of quality control products prepared with different dilutions at 37°C

[0057]

[0058] The results showed that after 14 days of thermal acceleration, the target and internal standard Ct changes of the diluent provided in Example 2 were significantly lower than those of the solutions in Comparative Examples 1 to 7 compared to day 0. This indicates that the quality control prepared with the diluent provided by the present invention has the highest stability compared to other diluents.

[0059] Test Case 3: Verification of Generality

[0060] Verification experiment of the universality of quality control products: The positive quality control product (Antu Yangkong) prepared in Example 2 of the present invention was used in duplicate with the quality control products of the test kits of three listed manufacturers, Fujian Taipu, Shanghai Zhijiang, and Jiangsu Shuoshi. The extraction and amplification reagents of the Taipu, Zhijiang, and Shuoshi test kits were used according to the instructions for extraction and amplification. The results are shown in Table 6. The positive quality control products of the Taipu, Zhijiang, and Shuoshi test kits can only be amplified and detected by their respective reagents and cannot be detected as positive by the other manufacturers; while the positive quality control product (Antu Yangkong) prepared by the present invention can be amplified as positive by all three manufacturers' test kits, indicating that this product has good universality.

[0061] Table 6 Verification of universality of quality control products from various manufacturers

[0062]

[0063] Test Example 4: Real-time stability evaluation experiment:

[0064] The prepared positive quality control product of Example 1, the supporting human β-globin gene plasmid and the negative quality control product prepared by the quality control product diluent, were placed in a 2-8°C environment for real-time stability assessment. Starting from day 0, the assessments were conducted for 0 / 2 / 4 / 6 / 9 / 12 / 14 / 18 months respectively. One sample of each negative quality control product and positive quality control product was extracted at each time point. Each sample was made in triplicate starting from nucleic acid extraction, and the sample was amplified and detected using fluorescent quantitative PCR. The results are shown in Tables 7, 8, and 9. The stability of the positive quality control products claimed in the kit instructions of the three listed manufacturers is that they need to be stored at -20°C for up to 12 months; while the negative quality control products and positive quality control products prepared by the present invention can be stably stored at 37°C for more than 14 days and stably stored at 2-8°C for at least 18 months, avoiding the influence of repeated freezing and thawing on the quality control results, which is significantly better than the three listed manufacturers. Moreover, the quality control preservation solution prepared by the present invention is significantly superior to the preservation solution for plasmids and pseudoviruses in patent application publication number CN110511926A, which claims that the solution can only be preserved for 9 months at 2-8°C.

[0065] Table 7 Storage stability of quality control products from various manufacturers

[0066] Listed test kit manufacturers Positive quality control composition Storage conditions Validity period Fujian Taipu Plasmid containing the target gene -20℃ 8 months Shanghai River / -20±5℃ 12 months Jiangsu Shuoshi Plasmid containing cfb gene fragment -20℃ 10 months

[0067] Table 8 Real-time stability of quality control products of the present invention

[0068]

[0069] Table 9 Real-time stability of quality control products of the present invention

[0070]

[0071] The Group B Streptococcus nucleic acid detection quality control provided by the present invention is consistent with actual GBS clinical swab samples and can be used as a standard quality control product for detection by marketed manufacturers. It can realistically simulate the in vitro GBS nucleic acid detection process and has high versatility, thus achieving comprehensive monitoring of GBS nucleic acid detection. Furthermore, the product has the advantages of low cost, high stability, and easy storage and transportation, making it suitable for large-scale industrial production. In summary, the present invention has important value and application prospects.

[0072] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A positive quality control product for detecting group B streptococci, characterized in that: Includes inactivated GBS strain, internal standard plasmid and diluent; The inactivated GBS strain includes at least one of types Ia, Ib, III, and V; The diluent consists of P300, P950, silicone, EDTA·2Na and HEPES buffer; The percentage of each component in the diluent is: 0.1~0.2vol% P300, 0.15~0.3vol% P950, 0.01~0.02vol% silicone defoamer and 0.035~0.07wt% EDTA·2Na.

2. The positive control product according to claim 1, characterized in that The mass fraction of each component in the dilution is: 0.1 vol% P300, 0.15 vol% P950, 0.01 vol% silicone, and 0.035 wt% EDTA·2Na; or, 0.2 vol% P300, 0.3 vol% P950, 0.02 vol% silicone, and 0.07 wt% EDTA·2Na.

3. The positive control product according to claim 1, characterized in that The internal standard plasmid is a plasmid containing the human β-globin gene.

4. The positive control product according to claim 3, characterized in that The human β-globin gene includes at least one gene with sequence ID MK476491.1, MK476483.1, and MK476464.

1.

5. The positive control product according to any one of claims 3 to 4, characterized in that The concentration of the GBS strain in the positive quality control product is 1×10 4 ~1×10 5 CFU / mL.

6. The positive control product according to any one of claims 1 to 4, characterized in that: The concentration of the internal standard plasmid containing human β-globin gene in the positive quality control product is 1×10 4 ~1×10 5 copies / mL.

7. The method for preparing the positive control product according to any one of claims 1 to 6, characterized in that: The following steps are involved: Activate, subculture, and expand the GBS strain; obtain a GBS strain suspension by centrifugation, resuspending, and inactivating; The GBS strain suspension and the internal standard plasmid solution containing the human β-globin gene were mixed, and P300, P950, silicone defoamer and EDTA·2Na were added to obtain a positive quality control product.

8. Use of the positive quality control product according to any one of claims 1 to 7 in the preparation of a nucleic acid detection kit for detecting group B streptococci.

Images