Method for detecting enterococcus faecalis in textile

Abstract

The invention relates to the technical field of textile detection, in particular to a method for detecting enterococcus faecalis in textiles, which comprises the following steps: S1, sample preparation: selecting a weighing method, a multi-point sampling elution method or a cotton swab smearing method according to the types of the textiles to prepare a sample homogenate; step S2, dilution and inoculation: performing 10-time series dilution on the sample homogenate, selecting a sample with 2-3 continuous dilution degrees, inoculating the sample to a bile aesculin sodium azide agar culture medium, and culturing at 36 + / -1 DEG C for 24-48 hours. According to the method, the detection technology of enterococcus faecalis in the textile is innovatively adopted, and the pollution condition of enterococcus faecalis in the textile is effectively monitored; the method has the following remarkable advantages that operation is easy and convenient, cost is low, complex instruments and equipment are not needed, the method is suitable for conventional detection of a primary laboratory, and the technology has outstanding originality and practicability in the aspect of improving the biological safety prevention and control capacity of the textile.

Description

Technical Field

[0001] The present invention relates to the technical field of textile detection, and more specifically, to a method for detecting Enterococcus faecalis in textiles. Background Art

[0002] Enterococcus faecalis is a robust species that can survive in important biological niches such as the human gastrointestinal tract and under strict environmental conditions, which facilitates their spread. They have a wide range of inherent drug resistances, are tolerant to the bactericidal activities of many fungicides, and are highly resistant to existing antibiotics, showing more and more virulence characteristics that promote colonization and infection. In addition, their ability to acquire new drug resistance and virulence determinants is also very developed, which poses a severe challenge to the biosecurity prevention and control of textiles.

[0003] Currently, Enterococcus faecalis has been detected in spring water, domestic drinking water, animal-derived foods, textiles, etc., and has the ability to resist acidic and alkaline and other inferior environments. It has been used as a hygiene indicator in water quality, cosmetics, and down and feather hygiene detection. There are already many industry standards for the biosecurity of imported and exported textiles in the textile hygiene inspection standards, which involve the total number of colonies, coliforms, Escherichia coli, Staphylococcus aureus, Candida albicans, etc. There have been relevant studies on the detection methods of Escherichia coli O157:H7, group B hemolytic streptococcus, Pseudomonas aeruginosa, and Salmonella in textiles, but the detection method of Enterococcus faecalis in textiles has been rarely studied. Therefore, it is very necessary and urgent to establish a detection method for Enterococcus faecalis in textiles.

[0004] For the differentiation of Enterococcus from other Gram-positive catalase-negative cocci by traditional culture detection methods, most Enterococcus are oxidase and catalase negative, salt-tolerant, bile-tolerant at 40%, hydrolyze esculin, and are able to grow in the presence of sodium azide. Enterococcus on blood agar can be α-, β-, or non-hemolytic, forming colonies of 1 mm - 2 mm in size, with a moist appearance. Based on their metabolic capabilities, there are different selective media on the market for isolating Enterococcus, and these selective media usually contain bile salts, sodium azide, antibiotics, and esculin or tetrazolium salts. Currently, there are commercial kits to standardize the detection of Enterococcus, but all require pre-isolation and culturing of isolates, and the detection period is relatively long. In addition, accurate differentiation between species cannot be achieved based solely on phenotypic testing. Its advantages lie in relatively simple operation, low cost, no need for complex instrument equipment, and the ability to obtain pure cultures, which is convenient for subsequent drug susceptibility tests and bacterial characteristic studies. However, this method also has obvious disadvantages, such as long detection time, being easily interfered by miscellaneous bacteria, and it is difficult to meet the diagnostic and rapid detection requirements for acute infections.

[0005] With the public's demand for high sensitivity, high specificity, and rapidity in detection technology, molecular biology methods such as PCR and quantitative fluorescence PCR have the advantages of high sensitivity, high specificity, and rapid detection. However, they have strict requirements for experimental conditions and techniques and are easily affected by contamination. Currently, more and more researchers are committed to combining traditional cultivation methods with automation to improve detection efficiency. Relatively new systems for Enterococcus classification and identification include matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), nucleic acid amplification tests (NAAT), peptide nucleic acid fluorescence in situ hybridization (PNA-FISH), and multilocus sequence typing (MLST). Identification based on MALDI-TOF MS is a powerful, rapid, and reliable method that has begun to be more widely used in routine testing in microbiology laboratories for species identification. MALDI-TOF MS has high sensitivity and can identify approximately 94% of isolates at the genus and species levels, including differentiating closely related genera and species; the NAAT method is based on PCR amplification and subsequent sequencing or array / hybridization or real-time PCR amplification of one or more genes, which can be used for microbial identification at the genus or species level and is also important for detecting antimicrobial resistance genes. In addition, 16S rRNA gene sequencing, PNA-FISH targeting species-specific rRNA, and multilocus sequence typing MLST can rapidly detect and identify strains. Recently, a new iteration for improving resolution has been achieved by performing core genome MLST (cgMLST), which is more cost-effective than whole genome sequencing WGS and average nucleotide difference analysis. These detection technologies have the advantages of high throughput, rapidity, and accuracy, and can simultaneously detect multiple microorganisms in a single experiment, greatly improving the detection efficiency. In the detection of Enterococcus faecalis, not only the presence of Enterococcus faecalis can be detected, but also its resistance genes, virulence genes, etc. can be analyzed, providing rich information for clinical treatment and epidemiological research. However, the high cost, great technical difficulty, and the need for professional equipment and technical personnel for operation and data analysis limit its application in some primary laboratories and its wide use in fields such as food, public health, and textiles.

[0006] Traditional cultivation detection methods have a long detection time and are easily interfered by miscellaneous bacteria, making it difficult to meet the needs for the diagnosis and rapid detection of acute infections. Moreover, the detection of Enterococcus faecalis by this method is still blank in the textile field.

[0007] Molecular biology methods such as PCR and quantitative fluorescence PCR have strict requirements for experimental conditions and techniques and are easily affected by contamination.

[0008] Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS), nucleic acid amplification test (NAAT), peptide nucleic acid fluorescence in situ hybridization (PNA-FISH), 16S rRNA gene sequencing, PNA-FISH targeting species-specific rRNA, multilocus sequence typing MLST, and core genome MLST (cgMLST) are limited in their application in some primary laboratories due to their high cost, great technical difficulty, and the need for professional equipment and technical personnel for operation and data analysis.

[0009] Therefore, we propose a method for detecting Enterococcus faecalis in textiles to solve the above problems. Summary of the Invention

[0010] To overcome the above defects of the prior art, an embodiment of the present invention provides a method for detecting Enterococcus faecalis in textiles to solve the problems raised in the above background art.

[0011] To achieve the above object, the present invention provides the following technical solution: A method for detecting Enterococcus faecalis in textiles, comprising the following steps:

[0012] Step S1: Sample preparation: Prepare a sample homogenate according to the type of textile by using the weighing method, multi-point sampling elution method, or cotton swab smearing method;

[0013] Step S2: Dilution and inoculation: Perform 10-fold serial dilution on the sample homogenate, select samples at 2-3 consecutive dilution levels and inoculate them on bile esculin azide agar medium, and culture at 36°C ± 1°C for 24-48 hours;

[0014] Step S3: Colony identification: Pick the typical colonies from grayish-brown to black on the PSE medium, and after purification, identify and confirm Enterococcus faecalis through Gram staining, microscopic examination, and biochemical tests;

[0015] Step S4: Result calculation and reporting: Calculate the concentration of Enterococcus faecalis in the sample according to the number of colonies and the dilution factor, and report it in the form of CFU / cm 2 、CFU / g or CFU / 100 cm 2 Form.

[0016] In a preferred embodiment, in step S1:

[0017] Weighing method: Open the submitted sample in a sterile manner, cut the sample evenly with sterile scissors, accurately weigh 25 g of the cut sample on an electronic balance, add it to a sterile homogenization bag with a full filter net containing 225 mL of sterile diluent after cutting it into pieces, and beat it with a beating homogenizer for 1 min - 2 min to fully mix and obtain a 1:10 sample homogenate.

[0018] In a preferred embodiment, in step S1:

[0019] Multi-point sampling elution method: Open the submitted sample in a sterile manner, evenly distribute 5 sampling points around and in the middle of the sample, and use a sterile template to cut according to an area range of 20 cm for each sampling point. 2 For every 20 cm 2 The sampling area is 1 test sample, and a total of 5 test samples are collected for each sample. The sampling area is 100 cm 2 , Put the above 5 collected test samples into a full-filter sterile homogenization bag containing 200 mL of sterile diluent, and beat with a beating homogenizer for 1 min - 2 min to fully mix and make a sample homogenate as the stock solution.

[0020] In a preferred embodiment, in step S1:

[0021] Cotton swab smearing method: Open the submitted sample in a sterile manner, moisten a sterile dry cotton swab with sterile diluent, evenly distribute 5 sampling points around and in the middle of the sample, and evenly smear according to an area range of 20 cm for each sampling point. Use 1 sterile dry cotton swab for each sampling point, immediately cut off the part of the cotton swab in contact with the hand with sterile scissors, and put the smeared part into a full-filter sterile homogenization bag containing 50 mL of sterile diluent to make a 1:10 sample homogenate. 2

[0022] In a preferred embodiment, in step S2, the bile esculin azide agar medium contains 0.1% - 0.5% bile salts and 0.05% - 0.1% esculin.

[0023] In a preferred embodiment, in step S2, the pH value of the bile esculin azide agar medium is 6.8 - 7.2, and the sterilization condition is autoclaving at 121 °C for 15 - 20 minutes.

[0024] In a preferred embodiment, in step S3, the colony morphology of the PSE medium is grayish-brown to black, round and convex, with a smooth surface, and the colony diameter is 1 mm - 2 mm.

[0025] In a preferred embodiment, after Gram staining, the catalase test is further used to exclude Staphylococcus bacteria.

[0026] In a preferred embodiment, in the biochemical test identification, the biochemical characteristics of Enterococcus faecalis are:

[0027] Ferment glucose, galactose, maltose;

[0028] Do not ferment erythritol, L-arabinose;

[0029] Produce arginine dihydrolase;

[0030] ​It does not produce catalase and β-galactosidase.

[0031] Technical effects and advantages of the present invention:

[0032] The method innovatively adopts the detection technology of Enterococcus faecalis in textiles, realizing the effective monitoring of the pollution situation of Enterococcus faecalis in textiles;

[0033] This method has the following significant advantages: simple operation, low cost, no need for complex instruments and equipment, suitable for routine detection in grass-roots laboratories, and this technology has outstanding originality and practicality in improving the biosafety prevention and control ability of textiles. Description of the drawings

[0034] Figure 1 It is a schematic diagram of the inspection process of Enterococcus faecalis in the present invention;

[0035] Figure 2 It is a schematic diagram of correlation analysis in the present invention;

[0036] Figure 3 It is a schematic diagram of analysis by the Bland-Altmen method in the present invention;

[0037] Figure 4 It is a schematic diagram of intermediate precision analysis in the present invention;

[0038] Figure 5 It is a schematic diagram of recovery analysis in the present invention;

[0039] Figure 6 It is a schematic diagram of reproducibility analysis in the present invention;

[0040] Figure 7 It is a correlation and linear regression fitting diagram of positive added samples in the present invention;

[0041] Figure 8 It is a diagram of the inter-laboratory verification results in the present invention. Detailed implementation manners

[0042] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] Refer to Figure 1-8 , a method for detecting Enterococcus faecalis in textiles;

[0044] 1 Sample preparation

[0045] 1.1 Weighing method

[0046] Open the submitted sample in a sterile manner, cut the sample evenly with sterile scissors, accurately weigh 25 g of the cut sample on an electronic balance, add it to a sterile homogenization bag with a full filter net containing 225 mL of sterile diluent after cutting into pieces, and beat it with a beating homogenizer for 1 min to 2 min to fully mix and obtain a 1:10 sample homogenate.

[0047] 1.2 Multi-point sampling elution method

[0048] Open the submitted sample in a sterile manner, evenly arrange 5 sampling points around and in the middle of the sample, and use a sterile template (20 cm 2 ) to cut according to an area range of 20 cm for each sampling point 2 , and take every 20 cm 2 of the sampling area as 1 test sample. A total of 5 test samples are collected for each sample, and the sampling area is 100 cm 2 . Put the 5 collected test samples into a sterile homogenization bag with a full filter net containing 200 mL of sterile diluent, beat it with a beating homogenizer for 1 min to 2 min, and fully mix to make a sample homogenate as the stock solution.

[0049] 1.3 Cotton swab smearing method

[0050] Open the submitted sample in a sterile manner, moisten a sterile dry cotton swab with sterile diluent, evenly arrange 5 sampling points around and in the middle of the sample, and evenly smear according to an area range of 20 cm 2 for each sampling point. Use 1 sterile dry cotton swab for each sampling point, immediately cut off the part of the cotton swab in contact with the hand with sterile scissors, and put the smeared part into a sterile homogenization bag with a full filter net containing 50 mL of sterile diluent to make a 1:10 sample homogenate.

[0051] 1.4 Selection of sample preparation method

[0052] Sample preparation generally takes the weighing method of adding 25 g of sample to 225 mL of diluent as the reference method. However, when the sample is large in area, thick or porous, multi-point sampling elution method should be used for sample preparation. When the following situations occur: a) The texture of the sample is dense and not easy to cut into pieces for preparation; b) The sample is relatively precious and the customer requires non-destructive testing, the cotton swab smearing method should be used for sample preparation.

[0053] When using the weighing method or multi-point sampling elution method, if the test sample absorbs a large amount of water and results in insufficient sample homogenate being drawn out, the amount of diluent can be increased in integer multiples until there is enough test sample homogenate.

[0054] When using the multi-point sampling elution method, if the test sample is too large or too small in area, the number of sampling points can be increased or decreased proportionally.

[0055] 2 Inspection Steps

[0056] 2.1 Inspection Process

[0057] The inspection process of Enterococcus faecalis is as Figure 1 shown;

[0058] 2.2 Dilution of Sample Homogenate

[0059] 2.2.1 Use a 1 mL sterile pipette or micropipette to aspirate 1 mL of the stock solution or sample homogenate, and slowly inject it along the wall of the tube into a sterile test tube containing 9 mL of sterilized diluent (note that the tip of the pipette or pipette tip should not touch the diluent surface). Shake the test tube or use another 1 mL sterile pipette to repeatedly pipette to make it evenly mixed, and prepare the sample homogenate of the next dilution degree.

[0060] 2.2.2 According to the estimation of the sample contamination situation, sequentially prepare a ten-fold increasing series of diluted sample homogenates. Each time of increasing dilution, change to another 1 mL sterile pipette or pipette tip. The whole process from preparing the sample homogenate to completing the sample inoculation shall not exceed 15 min.

[0061] 2.3 Inoculation and Incubation

[0062] According to the estimation of the contamination situation of the sample to be tested, select the sample homogenates of 2 - 3 consecutive appropriate dilution degrees. Respectively take 1 mL and add it into a sterile petri dish, and add 15 mL - 20 mL of PSE agar medium cooled to 46°C ± 1°C. Make 2 replicates for each dilution degree. At the same time, use 1 mL of sterilized diluent as a blank control. Gently rotate to mix well. After the medium solidifies, incubate it upside down at 36°C ± 1°C for 24 h - 48 h.

[0063] 2.4 Counting of Typical Colonies

[0064] 2.4.1 Counting should be carried out in a timely manner after the incubation is completed. It can be observed with the naked eye, and a magnifying glass or colony counter can be used when necessary. Colony counting is expressed in colony-forming units (CFU).

[0065] 2.4.2 Colony morphology: The typical or suspicious colonies of Enterococcus faecalis on the PSE agar plate are grayish-brown to black, round, convex, with a smooth surface and neat edges.

[0066] 2.4.3 Select the plates with the number of colonies between 25 CFU and 250 CFU, and respectively count the typical and suspicious Enterococcus faecalis colonies appearing on the plates. Record the specific number of colonies for the plates with the lowest dilution degree less than 25 CFU.

[0067] 2.5 Confirmatory Test

[0068] 2.5.1 Randomly select 5 or more typical or suspect colonies (select all if less than 5) on the PSE agar plate, streak-inoculate them onto the TSA plate respectively, incubate them in an inverted position at 36°C ± 1°C for 24 h ± 2 h, scrape the bacterial lawn for microscopic examination and biochemical identification.

[0069] 2.5.2 Perform Gram staining and microscopic examination. Enterococcus faecalis is a Gram-positive coccus, round or oval in shape, with a diameter of 0.5 μm - 1.0 μm, single or mostly in pairs or short chains, and without spores.

[0070] 2.5.3 Use a bacterial biochemical identification kit for biochemical identification, or a microbial biochemical identifier can also be used for biochemical identification. At the same time, set up negative control (blank) and positive control (ATCC29212 or BNCC186300). Enterococcus faecalis can ferment glucose, galactose, and maltose; it does not ferment erythritol or L-arabinose; it produces arginine dihydrolase; it does not produce catalase or β-galactosidase. The typical biochemical identification characteristics of Enterococcus faecalis are shown in Table 1;

[0071] Table 1 Biochemical identification characteristics of Enterococcus faecalis

[0072]

[0073] 2.6 Result calculation

[0074] 2.6.1 If:

[0075] a) Only one coefficient has the number of typical or suspect colonies on two plates between 25 CFU and 250 CFU, count the typical or suspect colonies on two plates of this dilution.

[0076] b) The number of typical or suspect colonies on two plates of the lowest dilution is less than 25 CFU, count the typical or suspect colonies on two plates of this dilution.

[0077] c) The number of colonies on all dilution plates is greater than 250 CFU and there are typical or suspect colonies, count the typical or suspect colonies on two plates of the highest dilution.

[0078] After the above typical or suspect colonies are confirmed, calculate according to formula (1):

[0079]

[0080] In the formula:

[0081] T - The number of Enterococcus faecalis colonies in the sample;

[0082] k - The coefficient obtained according to the sample preparation method, for the sample prepared according to 6.2, k = 2, for the samples prepared according to 6.1 and 6.3, k = 1;

[0083] A—the average number of typical or suspect colonies on two plates at a certain dilution;

[0084] B—the number of colonies positive in the confirmation test at a certain dilution;

[0085] C—the number of colonies used for the confirmation test in a certain test;

[0086] d—the dilution factor.

[0087] d) If the number of typical or suspect colonies on the plates of two consecutive dilutions is between 25 CFU and 250 CFU, then the typical or suspect colonies on these two consecutive dilution plates should all be counted. After colony confirmation, calculate according to formula (2):

[0088]

[0089] In the formula:

[0090] T—the number of Enterococcus faecalis colonies in the sample;

[0091] k—the coefficient obtained according to the sample preparation method. For the sample prepared according to 6.2, k = 2; for the sample prepared according to 6.1 and 6.3, k = 1;

[0092] A1—the average number of typical or suspect colonies on two plates at the first dilution;

[0093] B1—the number of colonies positive in the confirmation test at the first dilution;

[0094] A2—the average number of typical or suspect colonies on two plates at the second dilution;

[0095] B2—the number of colonies positive in the confirmation test at the second dilution;

[0096] C2—the number of colonies used for the confirmation test at the second dilution;

[0097] d—the dilution factor of the first dilution.

[0098] e) If there are no typical or suspect colonies on the plates of all dilutions, directly report the result as less than 1 multiplied by the lowest dilution multiple.

[0099] f) If there are other situations in the typical or suspect colony count, first calculate the two averages of the same dilution, select the result with the typical or suspect colony average within the appropriate counting range or close to the appropriate counting range for counting. If only one average is within the counting range, calculate according to formula (1) after confirmation; if both averages are within the counting range, calculate according to formula (2) after confirmation.

[0100] 3 Result reporting

[0101] Report the number of Enterococcus faecalis per square centimeter, per gram, or per 100 square centimeters of the sample, expressed as CFU / cm 2 , CFU / g, or CFU / 100 cm 2 . If the T value is 0, report it as less than 1 multiplied by the lowest dilution factor; if the T value is less than 100 CFU, report the actual value; if the T value is greater than or equal to 100 CFU, round the value according to the rules of GB / T 8170 during reporting and express it in exponential form of 10, with two significant figures retained.

[0102] This method has completed the in - laboratory verification of multiple parameters such as inclusivity, exclusivity, correlation analysis, relative accuracy, accuracy, precision, and linearity, and finally conducted an inter - laboratory collaborative verification.

[0103] Inclusivity and exclusivity analysis

[0104] Inclusivity refers to the detection ability of the method to be verified for the target microorganism, while exclusivity refers to the anti - interference ability of the method to be verified for non - target microorganisms

[107] .

[0105] All 30 target bacteria grew black colonies on the PSE plate and the surrounding medium turned brown, with typical colony morphology; after the biochemical characteristics were confirmed, they were all consistent with the added situation, indicating that the inclusivity of the above - established method meets the requirements.

[0106] Among the 32 added non - target bacteria, Enterococcus faecium, Enterococcus gallinarum, and Enterococcus durans also had typical black colonies that turned the surrounding medium brown on the PSE, but were confirmed as non - target bacteria in subsequent biochemical identifications; Staphylococcus aureus, Staphylococcus epidermidis, and Listeria monocytogenes all formed atypical colonies on the PSE plate, and the other non - target bacteria had no colony growth on the PSE plate. The detection results of the 32 added non - target bacteria were consistent with the added situation, and the established method has the ability to exclude the interference of non - target bacteria, indicating that the exclusivity technical indicators meet the requirements (Table 2).

[0107] Table 2 Exclusivity test results

[0108]

[0109]

[0110]

[0111] 2. Correlation

[0112] Based on the detection results of all samples, with the detection results (PSE count) of the method to be confirmed as the abscissa and the reference method (TSA plate count) as the ordinate, the Spearman correlation analysis was used to investigate the correlation between the two methods. The results are as Figure 2As shown, there is a strong positive correlation (r = 0.88) between the method to be confirmed and the reference method, and this correlation is extremely significant statistically (p < 0.0001).

[0113] 3. Relative accuracy

[0114] Based on the test results of all samples, calculate the mean and difference between the established method and the reference method. The bias can be estimated by the mean D of the differences in the measurement results of the established method, and the variation of the mean D can be described by the standard deviation SD of the differences. Using the mean as the abscissa and the difference as the ordinate, analyze using the Bland - Altman method. The results are as Figure 3 shown. The bias is -131.1, and the 95% LoA is -590.5 to 328.2. According to the distribution of the scatter plot, it can be seen that there are significant differences between the two test methods at high pollution levels, but the proportion of data points outside the 95% LoA line is small (4.4% < 5.0%), and most of the differences are within the 95% confidence interval of the bias, indicating that the established method meets the performance index requirements in terms of relative accuracy.

[0115] 4. Accuracy

[0116] Take the logarithm of the 5 parallel test results of each sample. The calculation formula for the β - expected tolerance interval (β - ETI) of the median value (Yi) of the test results of the established method is:

[0117]

[0118] The acceptable limit value AL of accuracy = ±0.5 logarithmic units

[107] . As can be seen from Table 3, the upper and lower limit values of the β - expected tolerance interval (β - ETI) of all test samples are within the acceptable limit range, indicating that the established method meets the performance index requirements in terms of accuracy.

[0119] Table 3 Results of accuracy analysis (CFU / g)

[0120]

[0121]

[0122] 5. Precision

[0123] (1) Repeatability

[0124] As shown in the results of Table 4, for the 3 types of substrates tested by the established method, with 6 samples for each type and each sample tested 5 times. The repeatability RSD ranges for the low, medium, and high contamination levels are 6.76% - 23.15%, 2.62% - 7.32%, and 6.04% - 19.52% respectively, all of which are less than 35%. Generally, the acceptable relative standard deviation (RSD) should not be greater than 35%, indicating that the precision of the established method is good. 2.62% - 7.32% < 10%, far less than the upper limit of the acceptable range, indicating that the number of colonies at the medium contamination level of the established method is within the appropriate counting range for plate counting.

[0125] Results of Precision Analysis in Table 4

[0126]

[0127] (2) Intermediate Precision

[0128] As can be seen from the results in item (4), there are no significant differences in the results among Group A, Group B, and Group C (P = 0.3169 > 0.05). At the same time, there are no significant differences in the test results of Group A, Group B, and Group C in the two batches of reagents (P values are 0.6747, 0.8988, and 0.2209 > 0.05 respectively), indicating that the changes in personnel, equipment, and reagent batches have little impact on the results, and there are no significant differences in the results, indicating that the stability and reliability of the newly established method are good.

[0129] From Figure 5 It can be seen that: compared with the true results of the contaminated bacterial solution, the recovery rates of the established method are all > 70% (73.3% - 130.7%). According to General Principles 9201, Volume IV of the Chinese Pharmacopoeia 2020 Edition, the recovery rate is greater than 70.0%

[109] , indicating a high degree of consistency between the test results and the true results, and the recovery rate of microorganisms meets the requirements, indicating that the accuracy index of the established method meets the requirements.

[0130] The RSD for the comparison of the test results of the two batches of culture media in Group A is 11.2%, the RSD for the comparison of the test results of the two batches of culture media in Group B is 12.1%, the RSD for the comparison of the test results of the two batches of culture media in Group C is 7.2%, and the RSD for the comparison of the results of the three groups is 13.2%. RSD < 35%, indicating that the intermediate precision of the newly established method meets the requirements and is not affected by personnel, equipment, and reagent batches.

[0131] (3) Reproducibility

[0132] High pollution level: The F value of the inter-laboratory comparison is 0.9972, which is greater than the F value of the matrix comparison, 0.7569, indicating that the influence of different laboratories on the test results is greater than that of the matrix type. From the test results of the two laboratories, it can be seen that for the three matrices of A cotton-polyester cloth, B cotton-linen cloth, and C polyester-ammonia, the RSDs of the test results of Laboratory 1 and Laboratory 2 are 2.1%, 3.7%, and 19.9% respectively, and P = 0.50 > 0.05, indicating that there is no difference in the test results of the two laboratories for the same matrix samples. The P value of the test results of different materials between laboratories is 0.48 > 0.05, indicating that there is no difference in the influence of different materials between laboratories on the test results.

[0133] Medium pollution level: The F value of the inter-laboratory comparison is 1.180, which is greater than the F value of the matrix comparison, 0.3987, indicating that the influence of different laboratories on the test results is greater than that of the matrix type. From the test results of the two laboratories, it can be seen that for the three matrices of A cotton-polyester cloth, B cotton-linen cloth, and C polyester-ammonia, the RSDs of the test results of Laboratory 1 and Laboratory 2 are 5.8%, 0.0%, and 27.3% respectively, and P = 0.46 > 0.05, indicating that there is no difference in the test results of the two laboratories for the same matrix samples. The P value of the test results of different materials between laboratories is 0.59 > 0.05, indicating that there is no difference in the influence of different materials between laboratories on the test results.

[0134] Low pollution level: The F value of the inter-laboratory comparison is 1.340, which is greater than the F value of the matrix comparison, 0.3507, indicating that the influence of different laboratories on the test results is greater than that of the matrix type. From the test results of the two laboratories, it can be seen that for the three matrices of A cotton-polyester cloth, B cotton-linen cloth, and C polyester-ammonia, the RSDs of the test results of Laboratory 1 and Laboratory 2 are 8.6%, 21.1%, and 13.3% respectively, and P = 0.43 > 0.05, indicating that there is no difference in the test results of the two laboratories for the same matrix samples. The P value of the test results of different materials between laboratories is 0.61 > 0.05, indicating that there is no difference in the influence of different materials between laboratories on the test results.

[0135] The RSDs of the three pollution levels are all less than 35%, and Enterococcus faecalis was not detected in the negative control samples of the two laboratories. The results show that the established method has good reproducibility.

[0136] 6 Linearity

[0137] The detection results of 6 positive spiked samples were analyzed by Spearman correlation analysis. As shown in Figure 7, the correlation coefficients r were -0.9747, -1.000, -1.000, -1.000, -1.000, -1.000 respectively, and the P values were all less than 0.05 and greater than 0.01, indicating that there was a significant negative correlation between the dilution factor and the detection results obtained by the established method. For the convenience of linear regression analysis, the logarithm of the dilution factor and the measured value were taken with base 10, and the linear regression equation of each sample was calculated. The range of the determination coefficient R2 was 0.95 - 0.98, and the P values were all < 0.0001. All points were within the 95% confidence interval, indicating that there was a good linear relationship between the dilution factor and the detection results. Moreover, there was no significant difference (P > 0.05) among the detection results of the 6 positive spiked samples, indicating that the established method had good repeatability.

[0138] 7 Inter-laboratory collaborative verification

[0139] Enterococcus faecalis was not detected in the negative control samples of each laboratory. As shown in Table 5, the relative standard deviations (RSD) of the detection results among laboratories for the three matrices of A cotton-polyester cloth, B cotton-linen cloth, and C polyester-ammonia at low pollution levels were 16.6%, 13.0%, and 17.2% respectively; the relative standard deviations (RSD) of the results among laboratories for the three matrices at medium pollution levels were 9.7%, 14.5%, and 13.7% respectively; the relative standard deviations (RSD) of the results among laboratories for the three matrices at high pollution levels were 7.9%, 4.0%, and 12.3% respectively. The RSDs of the three pollution levels were all less than 35%, and Enterococcus faecalis was not detected in the laboratory negative control samples.

[0140] Table 5 Analysis of inter-laboratory detection results

[0141]

[0142]

[0143] As Figure 8 shown, the letters in the figure indicate that there are significant differences between the two groups (P < 0.05). There were no significant differences (P > 0.05) in the detection results among laboratories for the three matrices of A cotton-polyester cloth, B cotton-linen cloth, and C polyester-ammonia at low, medium, and high pollution levels. In summary, the established method meets the requirements of inter-laboratory collaborative experiment verification, indicating that the established method is scientific and reliable.

[0144] Example 1

[0145] Sample preparation

[0146] (1) Weighing method

[0147] Open the submitted sample in a sterile manner, cut the sample evenly with sterile scissors, accurately weigh 25 g of the cut sample on an electronic balance, add it to a sterile homogenization bag with a full filter net containing 225 mL of sterile diluent after cutting it into pieces, and beat it with a beating homogenizer for 1 min to 2 min to fully mix it, obtaining a 1:10 sample homogenate.

[0148] (2) Multi-point sampling elution method

[0149] Open the submitted sample in a sterile manner, evenly arrange 5 sampling points around and in the middle of the sample, and use a sterile template (20 cm 2 ) to cut according to an area range of 20 cm for each sampling point 2 . Take every 20 cm 2 sampling area as 1 test sample, a total of 5 test samples are collected for each sample, and the sampling area is 100 cm 2 . Put the 5 collected test samples into a sterile homogenization bag with a full filter net containing 200 mL of sterile diluent, beat it with a beating homogenizer for 1 min to 2 min, and fully mix it to make a sample homogenate as the stock solution.

[0150] (3) Cotton swab smearing method

[0151] Open the submitted sample in a sterile manner, moisten a sterile dry cotton swab with sterile diluent, evenly arrange 5 sampling points around and in the middle of the sample, and evenly smear it according to an area range of 20 cm 2 for each sampling point. Use 1 sterile dry cotton swab for each sampling point, immediately cut off the part of the cotton swab in contact with the hand with sterile scissors, and put the smeared part into a sterile homogenization bag with a full filter net containing 50 mL of sterile diluent to make a 1:10 sample homogenate.

[0152] Example 2

[0153] Selection of sample preparation method

[0154] The sample preparation generally takes the method of adding 225 mL of diluent to 25 g of sample in accordance with Method 6.1 as the reference method. However, when the sample is large in area, thick, or porous, Method 6.2 should be used for sample preparation. When the following situations occur: a) The texture of the sample is dense and not easy to cut and prepare; b) The sample is relatively precious and the customer requests non-destructive testing, Method 6.3 should be used for sample preparation. When using Methods 6.1 and 6.2, if the tested sample absorbs a large amount of water and it is impossible to suck out enough sample homogenate, the amount of diluent can be increased in integer multiples until there is enough test sample homogenate. When using Method 6.2, if the area of the tested sample is too large or too small, the number of sampling points can be increased or decreased proportionally.

[0155] Example 3

[0156] Dilution of sample homogenate

[0157] (1) Using a 1 mL sterile pipette or micropipette (4.7), aspirate 1 mL of the stock solution or sample homogenate, and slowly inject it along the wall of the tube into a sterile test tube containing 9 mL of sterilized dilution solution (note that the tip of the pipette or pipette tip should not touch the dilution liquid surface). Shake the test tube or use a 1 mL sterile pipette to blow and beat repeatedly to mix evenly, and prepare the sample homogenate of the next dilution degree.

[0158] (2) According to the estimation of the sample contamination situation, successively prepare a ten-fold increasing series of diluted sample homogenates. Each time of increasing dilution, change to a new 1 mL sterile pipette or pipette tip. The whole process from preparing the sample homogenate to completing the sample inoculation shall not exceed 15 minutes.

[0159] Example 4

[0160] Inoculation and cultivation

[0161] According to the estimation of the contamination situation of the sample to be tested, select the sample homogenates of 2 - 3 consecutive appropriate dilution degrees. Respectively take 1 mL and add it into a sterile petri dish (4.8), and add 15 mL - 20 mL of PSE agar medium cooled to 46 °C ± 1 °C. Make 2 replicates for each dilution degree. At the same time, use 1 mL of sterilized dilution solution as a blank control. Gently rotate to mix thoroughly. After the medium solidifies, incubate it upside down at 36 °C ± 1 °C for 24 h - 48 h.

[0162] Example 5

[0163] Colony counting

[0164] (1) Counting should be carried out in a timely manner after the cultivation is completed. It can be observed with the naked eye, and a magnifying glass or colony counter can be used when necessary. Colony counting is expressed in colony-forming units (CFU).

[0165] (2) Colony morphology: Enterococcus faecalis forms colonies that are grayish brown to black, round, convex, with a smooth surface and neat edges on the PSE agar plate as typical or suspicious colonies.

[0166] (3) Select the plates with the number of colonies between 25 CFU and 250 CFU, and respectively count the typical and suspicious Enterococcus faecalis colonies appearing on the plates. Record the specific number of colonies for the plate with the lowest dilution degree less than 25 CFU.

[0167] Example 6

[0168] Confirmation experiment

[0169] (1) Randomly select 5 or more typical or suspicious colonies (select all if less than 5) on the PSE agar plate and streak inoculate them on the TSA plate, incubate upside down at 36 °C ± 1 °C for 24 h ± 2 h, scrape the bacterial lawn for microscopic examination and biochemical identification.

[0170] (2) Gram staining and microscopic examination. Enterococcus faecalis is a Gram-positive coccus, round or oval in shape, 0.5 μm - 1.0 μm in diameter, single, mostly in pairs or short chains, and without spores.

[0171] (3) Use a bacterial biochemical identification kit for biochemical identification, or a microbial biochemical analyzer can also be used for biochemical identification. At the same time, set up negative control (blank) and positive control (ATCC29212 or BNCC186300). Enterococcus faecalis can ferment glucose, galactose, and maltose; it does not ferment erythritol and L-arabinose; it produces arginine dihydrolase; it does not produce catalase and β-galactosidase.

[0172] Example 7

[0173] Result calculation

[0174] (1) Only when the number of typical or suspect colonies on two plates of one coefficient is between 25 CFU and 250 CFU, count the typical or suspect colonies on the two plates of this dilution.

[0175] (2) When the number of typical or suspect colonies on two plates of the lowest dilution is less than 25 CFU, count the typical or suspect colonies on the two plates of this dilution.

[0176] (3) When the number of colonies on all dilution plates is greater than 250 CFU and there are typical or suspect colonies, count the typical or suspect colonies on the two plates of the highest dilution.

[0177] After the above typical or suspect colonies are confirmed, calculate according to formula (1):

[0178]

[0179] In the formula:

[0180] T - The number of Enterococcus faecalis colonies in the sample;

[0181] k - The coefficient obtained according to the sample preparation method. For samples prepared according to 6.2, k = 2; for samples prepared according to 6.1 and 6.3, k = 1;

[0182] A - The average number of typical or suspect colonies on two plates of a certain dilution;

[0183] B - The number of colonies positive in the confirmation test of a certain dilution;

[0184] C - The number of colonies used for the confirmation test in a certain test;

[0185] d - The dilution factor.

[0186] (4) If the number of typical or suspect colonies on two consecutive dilution plates is between 25 CFU and 250 CFU, then all typical or suspect colonies on these two consecutive dilution plates should be counted. After colony confirmation, calculate according to formula (2):

[0187]

[0188] In the formula:

[0189] T—the number of Enterococcus faecalis colonies in the sample;

[0190] k—the coefficient obtained according to the sample preparation method. For samples prepared according to 6.2, k = 2; for samples prepared according to 6.1 and 6.3, k = 1;

[0191] A1—the average number of typical or suspect colonies on two plates of the first dilution;

[0192] B1—the number of colonies positive in the confirmation test of the first dilution;

[0193] A2—the average number of typical or suspect colonies on two plates of the second dilution;

[0194] B2—the number of colonies positive in the confirmation test of the second dilution;

[0195] C2—the number of colonies used for the confirmation test of the second dilution;

[0196] d—the dilution factor of the first dilution.

[0197] (5) If there are no typical or suspect colonies on the plates of all dilutions, directly report the result as less than 1 multiplied by the lowest dilution multiple.

[0198] (6) In case of other situations in the typical or suspect colony count, first calculate the two averages of the same dilution, select the result with the average number of typical or suspect colonies within or close to the appropriate counting range for counting. If only one average is within the counting range, calculate according to formula (1) after confirmation; if both averages are within the counting range, calculate according to formula (2) after confirmation.

[0199] Example 8

[0200] Result Report

[0201] Report the number of Enterococcus faecalis per square centimeter, per gram or per 100 square centimeters of the sample, in CFU / cm 2 、CFU / g or CFU / 100 cm 2Reporting. If the T value is 0, report it as less than 1 multiplied by the lowest dilution factor; if the T value is less than 100 CFU, report the actual value; if the T value is greater than or equal to 100 CFU, round the value according to the rules of GB / T 8170 during reporting and express it in exponential form of 10, with two significant figures retained.

[0202] This method innovatively adopts the detection technology of Enterococcus faecalis in textiles, realizing the effective monitoring of the pollution situation of Enterococcus faecalis in textiles. This method has the following significant advantages: simple operation, low cost, no need for complex instruments and equipment, and is suitable for routine detection in grass-roots laboratories. This technology has outstanding originality and practicality in enhancing the biosafety prevention and control ability of textiles.

[0203] This method can select three methods, namely the weighing method, the multi-point sampling and elution method, and the cotton swab smearing method, for sample preparation according to the type of experimental samples. After preparing the sample homogenate, it is serially diluted 10-fold and then inoculated onto bile esculin azide agar medium. After cultivation, suspicious colonies are obtained, and then inoculated onto tryptic soy agar medium to obtain purified colonies for Gram staining microscopy and eight biochemical identification tests, including glucose, galactose, maltose, erythritol, L-arabinose, arginine dihydrolase, catalase, and β-galactosidase. The advantages of this method are relatively simple operation, low cost, no need for complex instruments and equipment, can be widely applied in grass-roots laboratories, and can obtain pure cultures, which is convenient for subsequent drug sensitivity tests and bacterial characteristic studies.

[0204] Finally: The above are only the preferred embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting Enterococcus faecalis in textiles, characterized in that; It includes the following steps: Step S1: Sample preparation: Prepare a sample homogenate by selecting the weighing method, multi-point sampling and elution method, or cotton swab smearing method according to the type of textile; Step S2: Dilution and inoculation: Perform 10-fold serial dilutions on the sample homogenate, select samples at 2-3 consecutive dilution levels and inoculate them on bile esculin azide agar medium, and culture at 36°C ± 1°C for 24-48 hours; Step S3: Colony identification: Pick typical colonies ranging from grayish-brown to black on the PSE medium, purify them, and identify Enterococcus faecalis through Gram staining, microscopy, and biochemical tests; Step S4: Result calculation and reporting: Calculate the Enterococcus faecalis concentration in the sample based on the colony count and dilution factor, and report it in the form of CFU / cm 2 , CFU / g or CFU / 100 cm 2 for reporting.

2. The method for detecting Enterococcus faecalis in textiles according to claim 1, wherein: In step S1: Weighing method: Open the submitted sample in a sterile manner, evenly cut the sample with sterile scissors, accurately weigh 25 g of the cut sample on an electronic balance, cut it into pieces and add it to a full-filter sterile homogenization bag containing 225 mL of sterile diluent, and beat it with a beating homogenizer for 1 min - 2 min to fully mix and obtain a 1:10 sample homogenate.

3. The method for detecting Enterococcus faecalis in textiles according to claim 1, wherein: In step S1: Multi-point sampling elution method: Open the submitted sample in a sterile manner. Uniformly arrange 5 sampling points around and in the middle of the sample. Use a sterile template to cut according to an area range of 20 cm 2 for each sampling point. For every 20 cm 2 the sampling area is considered as 1 test sample. A total of 5 test samples are collected for each sample, and the sampling area is 100 cm 2 . Put the 5 collected test samples into a full-filter sterile homogenization bag containing 200 mL of sterile diluent, and beat with a beating homogenizer for 1 min to 2 min to fully mix and make a sample homogenate as the stock solution.

4. A method for detecting Enterococcus faecalis in textiles according to claim 1, characterized in that: In step S1: Cotton swab smearing method: Open the sample to be tested in a sterile manner. Moisten a sterile dry cotton swab with sterile diluent, and evenly arrange 5 sampling points around and in the middle of the sample. According to the sterile template, smear evenly within the area range of 20 cm for each sampling point. Use 1 sterile dry cotton swab for each sampling point. Immediately cut off the part of the cotton swab that has been in contact with hands with sterile scissors, and put the smeared part into a sterile homogenization bag with a full filter net containing 50 mL of sterile diluent to prepare a 1:10 sample homogenate. 2 For each sampling point, smear evenly within the area range of 20 cm. Use 1 sterile dry cotton swab for each sampling point. Immediately cut off the part of the cotton swab that has been in contact with hands with sterile scissors, and put the smeared part into a sterile homogenization bag with a full filter net containing 50 mL of sterile diluent to prepare a 1:10 sample homogenate.

5. A method for detecting Enterococcus faecalis in textiles according to claim 1, characterized in that: In step S2, the bile esculin azide agar medium contains 0.1% - 0.5% bile salts and 0.05% - 0.1% esculin.

6. A method for detecting Enterococcus faecalis in textiles according to claim 1, characterized in that: In step S2, the pH value of the bile esculin azide agar medium is 6.8 - 7.2, and the sterilization condition is autoclaving at 121°C for 15 - 20 minutes.

7. The method for detecting Enterococcus faecalis in textiles according to claim 1, characterized in that: In step S3, the colony morphology of the PSE medium is grayish-brown to black, round and convex, with a smooth surface, and the colony diameter is 1 mm - 2 mm.

8. A method for detecting Enterococcus faecalis in textiles according to claim 1, characterized in that: After Gram staining, further use the catalase test to exclude Staphylococcus bacteria.

9. A method for detecting Enterococcus faecalis in textiles according to claim 1, characterized in that: In the biochemical test identification, the biochemical characteristics of Enterococcus faecalis are: Ferment glucose, galactose, maltose; Do not ferment erythritol, L-arabinose; Produce arginine dihydrolase; Do not produce catalase and β-galactosidase.

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