Normal-temperature preservation solution for group B streptococcus in vaginal swab as well as preparation method and application of normal-temperature preservation solution

Abstract

The invention belongs to the technical field of microorganism preservation, and particularly relates to normal-temperature preservation liquid for group B streptococcus (GBS) in a vaginal swab as well as a preparation method and application of the normal-temperature preservation liquid. The invention provides a GBS normal-temperature preservation solution. The formula of the GBS normal-temperature preservation solution comprises glycerol, glucose, sodium citrate, EDTA, FBS, compound amino acid, NaCl and the like. The preservation solution ensures that GBS can be stably preserved for 30 days at normal temperature, the activity of GBS and the stability of RNA are effectively maintained, the problems of RNA degradation and bacterial activity loss in an existing preservation method are solved, and reliable sample support is provided for subsequent bacterial culture and RNA detection. The method is suitable for long-time preservation and subsequent detection of clinical vaginal swab samples, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial preservation technology, specifically relating to a room-temperature preservation solution for Group B Streptococcus in vaginal swabs, its preparation method, and its application. Background Technology

[0002] Group B Streptococcus (GBS), also known as agalactococcus, is an aerobic, Gram-positive streptococcus. As part of the normal human flora, GBS commonly colonizes the vagina and rectum of healthy women. However, as an opportunistic pathogen, GBS plays a significant role in perinatal medicine and has been identified as one of the main sources of maternal-to-child infection during the perinatal period. Epidemiological studies show that approximately 10% to 30% of pregnant women have vaginal GBS colonization, and 40% to 70% of these cases involve vertical transmission during delivery, leading to neonatal infection. Neonatal GBS infection can cause serious illnesses such as sepsis, meningitis, and pneumonia, with a mortality rate as high as 5% to 10%. Even among survivors, approximately 20% may suffer permanent neurological damage. With the widespread use of antibiotics, the problem of GBS resistance is becoming increasingly prominent. According to the 2024 China Antimicrobial Resistance Surveillance Network (CHINET) report, the resistance rates of GBS to levofloxacin, clindamycin, and erythromycin were 45.2%, 61.5%, and 75.7%, respectively. Given the serious consequences of GBS infection and the issue of drug resistance, the U.S. Centers for Disease Control and Prevention (CDC) issued the "Guideline for Perinatal GBS Prevention" as early as 2010, recommending routine screening for all pregnant women between 35 and 37 weeks of gestation. In 2021, China published the "Expert Consensus on the Prevention of Perinatal GBS," further emphasizing the importance of GBS screening in pregnancy management.

[0003] Currently, the main methods used clinically for GBS detection include bacterial culture, DNA detection, and RNA detection. Bacterial culture, the traditional "gold standard" for GBS detection, works by inoculating clinical samples onto selective media (such as sheep blood agar plates containing antibiotics) and culturing them at 35-37°C and 5% CO2 for 18-48 hours. Identification is then performed by observing characteristics such as β-hemolytic rings, Gram staining positivity, and a positive cAMP test. The main advantage of this method is its high specificity (nearly 100%) and the ability to simultaneously perform drug susceptibility testing. However, its significant drawbacks include: a long detection cycle (typically 24-72 hours), low sensitivity (approximately 60%-70%), strict requirements for sample collection and transportation conditions, and the inability to distinguish between colonizing and infectious bacteria. These limitations restrict its application in rapid clinical diagnosis, especially in neonatal infection cases requiring urgent intervention. In contrast, while DNA testing is faster (4 to 6 hours) and more sensitive than culture methods (detection limit up to 100 CFU / mL), its technical limitations are also significant: this method cannot distinguish between live and dead bacteria, and the results may include nucleic acid information from dead cells and apoptotic pathogens, thus failing to accurately reflect the current infection status of pregnant women or newborns. Clinical studies have shown that the false positive rate of DNA testing can be as high as 15% or more, and residual DNA can still be detected in the body after antibiotic use, for up to 3 weeks. These limitations significantly reduce the clinical guidance value of this testing method, thereby affecting the accurate formulation of antibiotic treatment plans.

[0004] In comparison, RNA detection technology exhibits significant advantages: firstly, as a product of gene expression, RNA detection results can directly reflect the active proliferation status of GBS in vivo; secondly, RNA has a short half-life (typically a few minutes), allowing for a more accurate reflection of the current infection status. However, RNA detection also faces technical challenges: GBS RNA is easily degraded in the in vitro environment after vaginal swab collection. Studies have shown that under normal conditions, the degradation rate of GBS RNA can exceed 90% within 5 minutes in vitro. This instability is mainly due to three factors: 1) altered bacterial activity leading to the termination of RNA synthesis; 2) rapid proliferation of other microorganisms in the sample (such as lactobacilli) causing a decrease in pH; and 3) RNase released after bacterial lysis accelerates RNA degradation.

[0005] Therefore, developing a vaginal swab-specific preservation solution that can effectively maintain the stability of GBS RNA has become a key technological bottleneck for achieving accurate detection, and is also a research direction that urgently needs to be broken through in the current GBS detection field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a vaginal swab preservation solution for Group B Streptococcus (GBS) preservation and its application method. This solution can effectively preserve the activity of GBS for a long time under normal temperature conditions and stabilize its RNA, thus solving the problems of RNA degradation and loss of bacterial activity in existing preservation methods.

[0007] This invention provides a room-temperature preservation solution for Group B Streptococcus, comprising components at the following concentrations: Glycerol 8-9 w / v %, glucose 1-2 w / v %, buffer salts 0.05-0.1 w / v %, metal chelators 0.02-0.05 w / v %, sodium chloride 0.9 w / v %, fetal bovine serum 35-60 w / v %, complex amino acids 0.005-0.03 w / v %.

[0008] Preferably, the concentration of glycerol is 8 w / v and the metal chelating agent is 0.02 w / v.

[0009] Preferably, it comprises components at the following concentrations: Glucose 1.2 w / v %, buffer salts 0.09 w / v %, metal chelators 0.02 w / v %, fetal bovine serum 50 w / v %, complex amino acids 0.02 w / v %.

[0010] Preferably, it consists of components with the following concentrations: Glycerol 8 w / v %, glucose 1.2 w / v %, buffer salts 0.09 w / v %, metal chelating agent 0.02 w / v %, sodium chloride 0.9 w / v %, fetal bovine serum 50 w / v %, complex amino acids 0.02 w / v %, and the balance being water.

[0011] Preferably, the buffer salt is selected from citrate, phosphate, or tris(hydroxymethyl)aminomethane hydrochloride; And / or, the metal chelating agent is selected from at least one of ethylenediaminetetraacetic acid (EDTA) and sodium EDTA. And / or, the complex amino acids comprise arginine, valine, cysteine, threonine, isoleucine, and histidine in a mass concentration ratio of 20:25:10:15:22:8.

[0012] Preferably, the pH of the room temperature preservation solution is 7.2 to 7.4.

[0013] The present invention provides a method for preparing a room temperature preservation solution as described in any of the preceding claims, comprising: mixing the components and adjusting the pH.

[0014] Preferably, the mixing step includes: sequentially adding glycerol, glucose, buffer salt, metal chelating agent, NaCl, and complex amino acids to water, stirring for 15-30 minutes, adding fetal bovine serum, and continuing to stir for 4-10 minutes; And / or, after adjusting the pH, the filter is sterilized.

[0015] This invention provides the use of the room temperature preservation solution as described in any of the preceding claims in the preparation of a kit for detecting Group B Streptococcus.

[0016] The present invention provides a kit comprising a room temperature storage solution as described in any of the preceding claims.

[0017] This invention provides a GBS (Guzmania ulmoides) preservation solution at room temperature by optimizing the types and concentration ratios of components. The solution comprises glycerol, glucose, sodium citrate, EDTA, FBS, complex amino acids, and NaCl. This preservation solution ensures stable preservation of GBS at room temperature (25°C ± 3°C) for up to 30 days, effectively maintaining GBS activity and RNA stability. It solves the problems of RNA degradation and loss of bacterial activity in existing preservation methods, and provides reliable sample support for subsequent bacterial culture and RNA detection. This method is suitable for long-term preservation and subsequent testing of clinical vaginal swab samples, showing promising application prospects.

[0018] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0019] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0020] Figure 1 Blood agar plates are shown for the preservation solution group and the group without preservation solution (containing only NaCl and sterile water) under the formulation of Example 1. Figure 2 Screening experiment for the types of components in the formula (glucose group (1.2%), maltose group (1.2%), ADP group (0.5mM), ATP group (0.5mM), total deficiency group (no energy negative control)); Figure 3 The effect of glycerol concentrations (5%, 6%, 7%, 8%, 9%, 10%) on GBS survival rate; Figure 4The effect of glucose concentrations (1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%) on GBS survival; Figure 5 The effect of sodium citrate concentrations (0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%) on GBS survival rate; Figure 6 The effect of EDTA concentrations (0.01%, 0.02%, 0.025%, 0.03%, 0.04%, 0.05%) on GBS survival rate; Figure 7 The effect of FBS concentrations (35%, 40%, 45%, 50%, 55%, 60%) on GBS survival rate; Figure 8 The effect of compound amino acid concentrations (0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%) on GBS survival rate; Figure 9 This is a graph showing the 30-day survival rate of GBS in the component-deficient control experiment. Detailed Implementation

[0021] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.

[0022] Example 1: A GBS room temperature preservation solution and GBS preservation method I. GBS room temperature preservation solution The GBS room temperature preservation solution in this embodiment has the following formulation: 8% glycerol, 1.2% glucose, 0.09% sodium citrate (trisodium citrate), 0.02% EDTA (ethylenediaminetetraacetic acid), 50% fetal bovine serum (FBS), 0.02% complex amino acids, 0.9% sodium chloride (NaCl), and sterile water (used to dissolve other components and adjust the overall concentration of the preservation solution). The complex amino acids are a combination of arginine (20%), valine (25%), cysteine ​​(10%), threonine (15%), isoleucine (22%), and histidine (8%) (purchased from Maclean's). The pH of the GBS preservation solution is 7.2–7.4.

[0023] The concentrations of all the components listed above are mass / volume concentrations (w / v, i.e., g / 100 mL).

[0024] II. Preparation method of GBS room temperature preservation solution The preparation method of GBS room temperature preservation solution is as follows: (1) Add glycerol, glucose, sodium citrate, EDTA, NaCl and compound amino acids to sterile water in the order of the formula, and stir on a magnetic stirrer for 20 minutes until completely dissolved; (2) Slowly add the preset amount of FBS and continue stirring for 5 minutes until the system is homogeneous; (3) Adjust the pH of the system to 7.2~7.4 using 1M HCl or 1M NaOH; (4) Sterilize by filtration through a 0.22μm filter membrane, dispense into sterile sealed centrifuge tubes (3mL per tube), seal and protect from light, and store at room temperature. Shelf life is 6 months.

[0025] III. GBS Room Temperature Storage Method (1) The sample was preserved as GBS bacterial culture. Mix 200 μL of GBS bacterial culture with 3 mL of preservation solution, vortex for 30 seconds to mix thoroughly; seal and store at room temperature (25℃±3℃) (no cold chain or light protection required).

[0026] During the storage period (0–30 days), 10 μL samples were taken at days 0, 7, 15, 22, and 30, spread onto blood agar plates, and incubated at 37°C with 5% CO2 for 48 hours. CFU and viability were then counted. Results are as follows: Figure 1 As shown, GBS can be stably stored for 30 days at room temperature (25℃±3℃). (See attached image) Figure 7 The survival rate reached 45.1% ± 3.2%, which solved the technical bottleneck of GBS activity loss in existing preservation methods and provided support for long-term transportation and subsequent testing of clinical vaginal swab samples.

[0027] (2) The sample was preserved as a vaginal swab. Vaginal secretions from the lower 1 / 3 of the vagina were collected using a nylon flocked swab and immediately placed into a centrifuge tube containing 1 mL of sterile saline. After vortexing for 1 minute to elute the swab, 200 μL of the eluent was mixed with 3 mL of the preservation solution at a volume ratio of 1:15, and then vortexed for 30 seconds to mix thoroughly. The centrifuge tube was then tightly capped and sealed with parafilm and stored at room temperature of 25℃±3℃.

[0028] In other embodiments, the glycerol concentration can be adjusted within the range of 8% to 9%, the glucose concentration can be adjusted within the range of 1% to 2%, the sodium citrate concentration can be adjusted within the range of 0.05% to 0.1%, the EDTA concentration can be adjusted within the range of 0.02% to 0.025%, the FBS concentration can be adjusted within the range of 35% to 60%, and the complex amino acid concentration can be adjusted within the range of 0.005% to 0.03%.

[0029] The technical solution of the present invention will be further explained through experiments below.

[0030] Experiment Example 1: Screening Experiment of Components in GBS Room Temperature Preservation Solution I. Experimental Methods 1. Experimental materials GBS clinical isolate (GBS-BJ01, type Ib), blood agar plates, glycerol, glucose, sodium citrate, EDTA, FBS, compound amino acids, NaCl (analytical grade), sterile water.

[0031] 2. Experimental Grouping (1) Screening experiment of the types of components in the formula GBS room temperature preservation solution was prepared according to the method in Example 1, wherein the 1.2% glucose was replaced with 1.2% maltose, 0.5mM ADP or 0.5mM ATP.

[0032] (2) Screening experiment on the concentration of components in the formula The experiments were grouped as shown in Table 1 to screen the optimal concentrations of each component in the formulation.

[0033] Table 1 (Only the target component is shown; other components are the same as in Example 1, n=3) (3) Component deficiency control experiment The experiments were grouped as shown in Table 2 to examine the effects of each component on the preservation effect of GBS.

[0034] Table 2 (1 mL system, n=3) 3. GBS bacterial culture preservation experiment at room temperature Mix 200 μL of GBS bacterial culture with 3 mL of GBS room temperature preservation solution, vortex for 30 seconds to mix thoroughly; seal and store at room temperature (25℃±3℃) (no cold chain or light protection required). During the storage period (0~30 days), take 10 μL samples at days 0, 7, 15, 22, and 30, spread them on blood agar plates, and incubate at 37℃ and 5% CO2 for 48 hours. Count CFU and viability.

[0035] II. Experimental Results (1) Results of screening experiments on the types of components in the formulation Experimental results are as follows Figure 2As shown, when glucose in the GBS storage solution was replaced with energy molecules such as maltose, ADP, or ATP, the survival rates of GBS varied significantly among the groups after 30 days of storage at room temperature (25℃±3℃): the glucose group achieved a survival rate of 45.1%, ensuring stable storage of GBS; while the survival rates of the maltose replacement group, ADP replacement group, and ATP replacement group were 5.0%, 3.8%, and 0.3%, respectively, with no statistically significant difference from the negative control group (0.6%) (P>0.05). This indicates that the type of energy molecule has a significant specific impact on the room temperature storage effect of GBS, and only when glucose is used as the energy molecule can GBS be stably preserved for 30 days under room temperature conditions.

[0036] (2) Results of screening experiments on the concentration of components in the formulation Experimental results are as follows Figures 3-8 As shown, the concentration of each component significantly affects the room temperature preservation effect of GBS, especially for glycerol, where the 8% concentration shows a significantly better room temperature preservation effect than other concentration groups; for EDTA, the 0.02-0.025% concentration shows a significantly better room temperature preservation effect than other concentration groups. The results of this experiment indicate that the optimal formulation of the vaginal swab GBS room temperature preservation solution of this invention is: 8% glycerol, 1.2% glucose, 0.09% sodium citrate, 0.02% EDTA, 50% FBS, 0.02% compound amino acids, and 0.9% NaCl.

[0037] (3) Results of the component missing control experiment Experimental results are as follows Figure 9 As shown, the results indicate that at 30 days, the survival rate of the experimental group (45.1%) was 75 times that of the total deficiency group (0.6%) and 3.2 times that of the lowest single deficiency group (EDTA deficiency group 14.2%); and the survival rate of the compound amino acid deficiency group (30.3%) was 32.8% lower than that of the experimental group. The synergy index was calculated using the synergy effect multiple: synergy multiple of compound amino acids = 45.1% ÷ 30.3% ≈ 1.49, meaning that the overall effect of the formula was improved by 49% after the addition of compound amino acids. Furthermore, the survival rate of the compound amino acid deficiency group (30.3%) was significantly higher than the average of other single deficiency groups (20.0%), demonstrating its ability to buffer the functional deficiencies of other components. This indicates that compound amino acids have a synergistic effect with other components, highlighting the technical value of the component combination in this formula.

[0038] The above results demonstrate that, through screening experiments based on the presence, type, and concentration of components, the optimal formulation of the vaginal swab GBS room temperature preservation solution of this invention is: 8% glycerol, 1.2% glucose, 0.09% sodium citrate, 0.02% EDTA, 50% FBS, 0.02% compound amino acids, and 0.9% NaCl. This formulation, through the synergistic effect of its components, can stably preserve GBS for 30 days at room temperature (25℃±3℃). Figure 1 The survival rate reached 45.1% ± 3.2%, which solved the technical bottleneck of GBS activity loss in existing preservation methods and is suitable for long-term transportation and subsequent testing of clinical vaginal swab samples.

[0039] As can be seen from the above embodiments and experimental examples, this invention provides a preservation solution for GBS transport and storage at room temperature, its preparation method, and its application. This invention provides a GBS room-temperature preservation solution by optimizing the types and concentration ratios of components. The formulation includes glycerol, glucose, sodium citrate, EDTA, FBS, complex amino acids, NaCl, and other ingredients. The preservation solution of this invention ensures that GBS can be stably preserved at room temperature (25°C ± 3°C) for up to 30 days, effectively maintaining the activity and RNA stability of GBS. It solves the problems of RNA degradation and loss of bacterial activity in existing preservation methods and provides reliable sample support for subsequent bacterial culture and RNA detection. This method is suitable for long-term preservation and subsequent detection of clinical vaginal swab samples and has promising application prospects.

Claims

1. A room-temperature preservation solution for Group B Streptococcus, characterized in that, It includes components at the following concentrations: Glycerol 8-9 w / v %, glucose 1-2 w / v %, buffer salts 0.05-0.1 w / v %, metal chelators 0.02-0.05 w / v %, sodium chloride 0.9 w / v %, fetal bovine serum 35-60 w / v %, complex amino acids 0.005-0.03 w / v %.

2. The room temperature preservation solution according to claim 1, characterized in that: The concentration of glycerol was 8 w / v and the concentration of metal chelating agent was 0.02 w / v.

3. The room temperature preservation solution according to claim 1 or 2, characterized in that, It includes components at the following concentrations: Glucose 1.2 w / v %, buffer salts 0.09 w / v %, metal chelators 0.02 w / v %, fetal bovine serum 50 w / v %, complex amino acids 0.02 w / v %.

4. The room temperature preservation solution according to claim 3, characterized in that, It consists of components at the following concentrations: Glycerol 8 w / v %, glucose 1.2 w / v %, buffer salts 0.09 w / v %, metal chelating agent 0.02 w / v %, sodium chloride 0.9 w / v %, fetal bovine serum 50 w / v %, complex amino acids 0.02 w / v %, and the balance being water.

5. The room temperature preservation solution according to claim 1, characterized in that: The buffer salt is selected from citrate, phosphate, or tris(hydroxymethyl)aminomethane hydrochloride; And / or, the metal chelating agent is selected from at least one of ethylenediaminetetraacetic acid (EDTA) and sodium EDTA. And / or, the complex amino acids comprise arginine, valine, cysteine, threonine, isoleucine, and histidine in a mass concentration ratio of 20:25:10:15:22:

8.

6. The room temperature preservation solution according to claim 1, characterized in that: The pH of the room temperature preservation solution is 7.2~7.

4.

7. The method for preparing the room temperature preservation solution according to any one of claims 1-6, characterized in that, It includes: The components are mixed and the pH is adjusted.

8. The preparation method according to claim 7, characterized in that, The mixing steps include: sequentially adding glycerol, glucose, buffer salt, metal chelating agent, NaCl, and complex amino acids to water, stirring for 15-30 minutes, adding fetal bovine serum, and continuing to stir for 4-10 minutes; And / or, after adjusting the pH, the filter is sterilized.

9. The use of the room temperature preservation solution according to any one of claims 1-6 in the preparation of a kit for detecting Group B Streptococcus.

10. A reagent kit, characterized in that: It includes the room temperature preservation solution as described in any one of claims 1-6.

Images