Chlamydia trachomatis detection capability verification item, preparation method and laboratory application thereof

Abstract

The present invention relates to a proficiency testing article for Chlamydia trachomatis detection, its preparation method, and laboratory application. The proficiency testing article is obtained by culturing CT serotype E in a CT-sensitive cell line and includes a lyophilized preparation and a reconstituted solution. The article simulates clinical CT samples and serves as a proficiency testing article for clinical CT infection diagnosis using colloidal gold, latex, and nucleic acid detection methods. The article is used for external quality assessment, internal quality control, and kit performance evaluation in clinical laboratories.

Description

Technical Field

[0001] The present invention belongs to the field of medical testing, and in particular relates to a Chlamydia trachomatis detection capability verification article, a preparation method thereof, and laboratory application thereof. Background Art

[0002] Chlamydia trachomatis (CT) is a tiny intracellular parasitic microorganism that is divided into mouse biotype, trachoma biotype and lymphogranuloma venereum biotype. The latter two are related to human diseases and are common infectious microorganisms of sexually transmitted diseases. They can cause human urogenital tract infections, such as urethritis and epididymitis in men, and cervicitis and intrauterine infection in women. Reproductive tract infection with CT in pregnant women can lead to pneumonia and ophthalmitis in neonates.

[0003] Currently, common methods for rapid clinical diagnosis of CT infection include colloidal gold, latex, and molecular diagnostic techniques based on nucleic acid detection. Existing commercial CT proficiency test items are mostly crude CT protein extracts, which are expensive to prepare, have low protein concentrations and antigen titers, and fail to cover high, medium, and low concentration levels. Furthermore, liquid proficiency test items prepared using protein extraction methods are prone to protein denaturation, have poor stability, and a short shelf life. When tested using colloidal gold, positive results are only displayed for a short time and easily fade. Furthermore, protein extract proficiency test items are not compatible with nucleic acid detection diagnostic techniques, while CT proficiency test items prepared using plasmid and gene amplification technologies are only compatible with nucleic acid detection diagnostic techniques and not with colloidal gold or latex methods. The low quality and short shelf life of the proficiency test items used in test kits not only complicate laboratory quality control but also fail to guarantee test quality and safety. This leads to missed and incorrect detections during clinical screening for CT infection, leading to misdiagnosis. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a Chlamydia trachomatis detection proficiency testing item, its preparation method and laboratory application, and successfully prepare a CT proficiency testing item that can effectively simulate clinical samples, and prove that it can be used for external quality assessment of clinical laboratories.

[0005] The invention provides a Chlamydia trachomatis detection proficiency verification item, which is obtained by culturing CT serotype E in a CT sensitive cell line and comprises a lyophilized preparation and a reconstituted solution thereof.

[0006] The freeze-dried preparation of the proficiency test item is CT bacteria that are inactivated after culture, enrichment, purification, and are obtained by vacuum freeze-drying; the reconstituted solution of the proficiency test item is a PBS solution containing 0.45% sodium chloride, with a pH range of 6.8 to 7.2.

[0007] The CT-sensitive cell line includes at least one of HELA-299 cells, HaCaT cells, McCoy cells, and BGMK cells.

[0008] When in use, the concentration of the proficiency test item is adjusted by adding different volumes of reconstitution solution to the lyophilized preparation, corresponding to different CT concentrations in clinical samples, covering high, medium and low concentrations.

[0009] The present invention also provides a method for preparing Chlamydia trachomatis proficiency testing items, which mainly comprises the following steps:

[0010] The CT-sensitive cell line is cultured in a 6-well plate, and after growing into a monolayer of cells, the cell culture medium is removed and a cell culture medium containing cycloheximide is added; CT serotype E is then inoculated, and after completion of the inoculation, the cells are centrifuged and cultured, and the growing CT inclusion bodies are observed. The infected cells are scraped off with a cell brush, ultrasonically disrupted, and centrifuged to remove cell debris, and the CT is then centrifuged to precipitate the CT; finally, the cells are suspended in PBS, inactivated, aliquoted, freeze-dried, and stored at low temperature for later use to obtain a lyophilized powder for Chlamydia trachomatis proficiency testing items; before use, the lyophilized powder is removed, equilibrated to room temperature, and then dissolved in a reconstitution solution.

[0011] The present invention also provides a laboratory application of Chlamydia trachomatis proficiency testing items.

[0012] The applications include clinical laboratory external quality assessment, internal quality control and test kit performance evaluation.

[0013] The application methods include colloidal gold method, latex method and molecular diagnostic technology experimental method based on nucleic acid detection.

[0014] Beneficial effects

[0015] (1) The present invention successfully prepared CT proficiency testing items that can simulate clinical samples. Based on CT culture, cell lysis, centrifugal enrichment, CT inactivation, freeze-drying, and reconstitution, the main preparation process is mature, low-cost, and easy to transfer.

[0016] (2) The present invention is based on CT culture, which is easy to mass-produce. The concentration of proficiency testing items can be effectively controlled and divided into three concentration levels: high, medium, and low. The similarity with actual clinical samples is high;

[0017] (3) The preparation process of the present invention effectively protects the surface antigen structure and nucleic acid components of CT and is applicable not only to the colloidal gold method and the latex method, but also to molecular diagnostic technology based on nucleic acid detection, with good sensitivity and specificity;

[0018] (4) The present invention inactivates CT, reducing biosafety risks during transportation and experimental operation. The present invention replaces sucrose phosphate buffer with a PBS solution with a pH of 6.8 to 7.2 and containing 0.45% sodium chloride as a buffer for removing cell debris after CT culture by centrifugation. At the same time, it also serves as a reconstitution solution for the finished product (dry powder preparation) of the proficiency test article.

[0019] (5) The dry powder preparation in the finished product of the proficiency test article of the present invention has good water solubility and is easy to operate. The reconstituted solution provides the optimal pH and ionic medium environment required for the colloidal gold method and latex method, making the experimental results more reliable and stable.

[0020] (6) The finished products of the proficiency testing items of the present invention are dry powder preparations and reconstituted solutions, which are convenient to store, have a long shelf life, and have good uniformity and stability. The present invention has a wide range of clinical laboratory application value. It is not only suitable for clinical laboratory external quality assessment to evaluate the laboratory's inspection quality, but also can be packaged and prepared into CT proficiency testing item kits for indoor quality control of CT detection reagents and evaluation of the sensitivity and specificity of the detection kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of the CT PCR fluorescent probe method in Example 1 are as follows;

[0022] Figure 2 The CT colloidal gold method and latex method test results of Example 1 are shown below:

[0023] Figure 3 The results of the CT PCR fluorescent probe method in Example 2 are as follows;

[0024] Figure 4 These are the test results of the CT colloidal gold method and latex method in Example 2. DETAILED DESCRIPTION

[0025] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0026] Example 1

[0027] This example takes the HELA-299 cell line as an example:

[0028] 1.1 Cell lines and strains

[0029] HELA-299 cell lines were used for Chlamydia trachomatis culture and were purchased from Wuhan Punosai Life Science Co., Ltd. CT serotype E was from the American type culture collection (ATCC) and purchased from Beina Chuanglian Biotechnology Co., Ltd.

[0030] 1.2 Reagents

[0031] RPMI-1640 medium (GIBCO), fetal bovine serum (GIBCO), CT nucleic acid detection kit (QIAGEN), and anti-CT monoclonal antibody (MicroTrak) were purchased from Shanghai Shiyan Biotechnology Co., Ltd. CT antigen detection kit (colloidal gold method), CT antigen detection kit (latex method), and cycloheximide (Sigma) were purchased from Shanghai Kaichuang Biotechnology Co., Ltd.

[0032] 1.3 Instruments

[0033] INC 153 carbon dioxide incubator (Memmert); NU-430-400E class II CO2 physical safety cabinet (Nuaire); 2-16P laboratory universal desktop centrifuge (Sartorius); QuantStudio 5 fluorescence quantitative PCR instrument (Thermo Fisher); SCIENTZ-ⅡD ultrasonic cell disruptor (Xinzhi); freeze dryer (LABCONCO).

[0034] 1.2 Methods

[0035] 1.2.1 Cell culture ELA-299 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator and passaged three times a week. After about 2 weeks, cells in good growth condition were obtained for use.

[0036] 1.2.2 CT Culture HELA-299 cells were grown in 6-well plates. After growth into a monolayer, the cell culture medium was removed and a cell culture medium containing 2 g / mL cycloheximide was added. CT serotype E was then inoculated. After inoculation, the cells were centrifuged at 1600 rpm at 35°C for 60 min. After centrifugation, the cells were incubated at 37°C with 5% CO2 for 72 h. After incubation, the growth of CT inclusion bodies was observed using an inverted microscope. Infected cells were scraped off with a cytobacterial brush and disrupted with ultrasound. Cell debris was removed by centrifugation at 1600 rpm for 10 min, and the CT was precipitated by high-speed centrifugation at 1600 rpm for 10 min. The cells were resuspended in PBS, inactivated in a water bath at 60°C for 30 min, and stored until use.

[0037] 1.2.3 Determination of CT Concentration HELA-299 cells were grown in a 6-well cell culture plate with a 24 mm diameter coverslip. The culture medium was discarded and a cell culture medium containing 2 g / mL cycloheximide was added. CT serotype E was inoculated and centrifuged at 2400 rpm and 35°C for 60 min. The cells were incubated at 37°C and 5% CO2 for 48 h. The culture medium was discarded, the cells were washed three times with PBS, and the coverslips were fixed with methanol for 10 min. The coverslips were stained with direct fluorescence using an anti-CT monoclonal antibody. The number of inclusion bodies was counted under a fluorescence microscope and the concentration of the prepared CT serotype E was calculated to be 3.50 × 10 9 IFU / mL.

[0038] 1.2.4 Sample Preparation for the External Quality Assessment Survey: CT suspended in PBS solution was dispensed into 1ml cryovials, 0.5ml per vial. The vials were frozen at -70°C overnight. The frozen vials were removed and placed in a LABCONCO freezer for lyophilization for at least 10 hours. After lyophilization, the vials were capped, labeled with the bacterial name and batch number, and stored in a freezer. Based on the objectives of the external quality assessment, five reconstitution solutions (PBS containing 0.45% sodium chloride, adjusted to a pH range of 6.8-7.2) and five proficiency testing items were prepared. Two of these were freeze-dried powders of cell culture fluid not inoculated with CT as negative samples, and three were freeze-dried powders of the prepared CT. The samples were randomly numbered and stored for future use. As needed, the reconstitution solution was added at ratios of 1:1, 1:5, and 1:10 to produce high, medium, and low concentrations of CT proficiency testing items. These can be used for nucleic acid testing and antigen testing kits.

[0039] 1.2.5 Verification of Uniformity and Stability of External Quality Assessment Products and Simultaneous Stability Testing: Ten CT proficiency testing items were tested for uniformity, and ten for stability (repeated after 1 week, 4 weeks, 8 weeks, and 12 weeks, respectively; see Table 1). After the proficiency testing items were distributed, three additional items were tested for simultaneous stability testing. The reagents used were CT antigen detection kits (colloidal gold / latex methods), and the verification results all met the requirements.

[0040] Table 1 Results of stability tests of external quality control products

[0041]

[0042]

[0043] 1.2.6 Inter-laboratory quality assessment survey project for CT testing in Shanghai Currently, there are 97 laboratories in Shanghai that carry out CT testing projects, of which 54 laboratories are participating in this inter-laboratory quality assessment survey plan. Samples are sent to participating laboratories via cold chain transportation, and they are required to use their laboratory routine detection kits and methods for testing within the specified time. The test results will be reported. The score of each laboratory will be calculated based on the conformity of the reported test results with the expected results. Qualification judgment principle: 20 points will be awarded for each correct identification, and a total score of ≥80 points will be considered qualified. The scores will be summarized, and statistical analysis will be conducted on erroneous test results. Antigen test kits will be used for all inter-laboratory quality assessment survey projects.

[0044] 1.3 Statistical methods

[0045] Data were analyzed using EXCEL 2007. Enumeration data were expressed as cases or rates.

[0046] 2 Results

[0047] 2.1 CT nucleic acid testing

[0048] The results of CT PCR-fluorescent probe method are shown in Figure 1 .

[0049] 2.2 CT antigen reagent detection

[0050] CT proficiency testing item test results, see Figure 2 The results of the CT antigen detection kit colloidal gold method and latex method were consistent.

[0051] 2.3 Laboratory Report

[0052] The collected and reported results are shown in Tables 2 and 3. Samples were distributed to 54 laboratories, and 51 data sets were collected. Of these, 22 laboratories used the colloidal gold method and 29 used the latex method. The results were all in line with expectations.

[0053] Table 2 Results of CT proficiency testing items survey (colloidal gold method)

[0054] [Number of laboratories (%)]

[0055]

[0056] Table 3 CT proficiency testing item survey results (latex method)

[0057] [Number of laboratories (%)]

[0058]

[0059]

[0060] Example 2

[0061] This example takes the McCoy cell line as an example:

[0062] 1 Materials and Methods

[0063] 1.1 Cell lines and strains

[0064] McCoy cells were used for Chlamydia trachomatis culture and were purchased from Wuhan Punosai Life Science Co., Ltd. CT serotype E was from the American type culture collection (ATCC) and purchased from Beina Chuanglian Biotechnology Co., Ltd.

[0065] 1.2 Reagents

[0066] MEM medium (GIBCO), fetal bovine serum (GIBCO), CT nucleic acid detection kit (QIAGEN), and anti-CT monoclonal antibody (MicroTrak) were purchased from Shanghai Shiyan Biotechnology Co., Ltd. CT antigen detection kit (colloidal gold method), CT antigen detection kit (latex method), and cycloheximide (Sigma) were purchased from Shanghai Kaichuang Biotechnology Co., Ltd.

[0067] 1.3 Instruments

[0068] INC 153 carbon dioxide incubator (Memmert); NU-430-400E class II CO2 physical safety cabinet (Nuaire); 2-16P laboratory universal desktop centrifuge (Sartorius); QuantStudio 5 fluorescence quantitative PCR instrument (Thermo Fisher); SCIENTZ-ⅡD ultrasonic cell disruptor (Xinzhi); freeze dryer (LABCONCO).

[0069] 1.2 Methods

[0070] 1.2.1 Cell culture McCoy cells were cultured in MEM medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator and passaged three times a week. After about 2 weeks, cells in good growth condition were obtained for use.

[0071] 1.2.2 CT Culture: McCoy cells were grown in 6-well plates. After growth into a monolayer, the cell culture medium was removed and cell culture medium containing 2 g / mL cycloheximide was added. CT serotype E was then inoculated. After inoculation, the cell culture plates were centrifuged at 1600 rpm at 35°C for 60 minutes. After centrifugation, the plates were incubated at 37°C with 5% CO2 for 72 hours. After incubation, the growth of CT inclusion bodies was observed using an inverted microscope. Infected cells were scraped off with a cytobacterial brush and disrupted by ultrasonication. Cell debris was removed by centrifugation at 1600 rpm for 10 minutes. The CT was then pelleted by high-speed centrifugation at 1600 rpm for 10 minutes. The cells were resuspended in PBS, inactivated in a water bath at 60°C for 30 minutes, and stored until ready for use.

[0072] 1.2.3 Determination of CT Concentration McCoy cells were grown in 6-well cell culture plates with 24 mm diameter coverslips. The culture medium was discarded and cell culture medium containing 2 g / mL cycloheximide was added. CT serotype E was inoculated, centrifuged at 2400 rpm and 35°C for 60 min, and incubated at 5% CO2 and 37°C for 48 h. The culture medium was discarded, the cells were washed three times with PBS, and the coverslips were fixed with methanol for 10 min. The coverslips were stained with direct fluorescence using an anti-CT monoclonal antibody. The number of inclusion bodies was counted under a fluorescence microscope, and the concentration of the prepared CT serotype E was determined to be 2.98 × 10 9 IFU / mL.

[0073] 1.2.3 Preparation of CT Quality Control: Dispense CT suspended in PBS into 1ml cryovials, 0.5ml per vial. Freeze the vials in a -70°C freezer overnight. Remove the frozen vials and place them in a LABCONCO freeze dryer for at least 10 hours. After lyophilization, remove the vials, cap them, label them with the bacterial name and batch number, and store them in a freezer. As needed, reconstitute the vials with reconstitution solution at ratios of 1:1, 1:5, and 1:10 to produce high, medium, and low concentration CT proficiency testing materials. These can be used for nucleic acid testing and antigen testing kits.

[0074] 1.2.4 Homogeneity and Stability Verification of CT Proficiency Test Items: 10 CT proficiency test items were tested for homogeneity, and 10 for stability (repeated after 1 week, 4 weeks, 8 weeks, and 12 weeks, respectively; see Table 4). The reagents used were CT antigen detection kits (colloidal gold / latex methods), and the verification results met the requirements.

[0075] Table 4 CT proficiency test results of items stability

[0076]

[0077] 2.1 CT nucleic acid testing

[0078] The results of CTPCR-fluorescence probe method are shown in Figure 3 .

[0079] 2.2 CT antigen reagent detection

[0080] CT antigen test results are shown in Figure 4 The results of the CT antigen detection kit colloidal gold method and latex method were consistent.

Claims

1. A Chlamydia trachomatis detection capability verification article, characterized by: The proficiency testing items are obtained by culturing CT serotype E with a CT-sensitive cell line, including lyophilized preparations and reconstituted solutions thereof; The preparation method of the proficiency testing item comprises the following steps: culturing a CT-sensitive cell line in a 6-well plate, removing the cell culture medium after the cells grow into a monolayer, and adding a cell culture medium containing cycloheximide; then inoculating CT serotype E, centrifuging and culturing after the inoculation is completed, observing the grown CT inclusion bodies, scraping the infected cells with a cell brush, ultrasonically disrupting them, centrifuging to remove cell debris, and then centrifuging to precipitate the CT; finally, suspending the cells in a PBS solution containing 0.45% sodium chloride with a pH range of 6.8-7.2, inactivating the cells, packaging the cells, freeze-drying them, and storing them at low temperature for later use to obtain a freeze-dried powder of the Chlamydia trachomatis proficiency testing item; and before use, removing the freeze-dried powder, equilibrating the cells to room temperature, and then dissolving the powder in a reconstitution solution.

2. The proficiency testing article according to claim 1, characterized in that: The freeze-dried preparation of the proficiency test item is CT bacteria that are inactivated after culture, enrichment, purification, and are obtained by vacuum freeze-drying; the reconstituted solution of the proficiency test item is a PBS solution containing 0.45% sodium chloride, with a pH range of 6.8 to 7.

2.

3. The proficiency testing article according to claim 1, characterized in that: The CT-sensitive cell line includes at least one of HELA-299 cells, HaCaT cells, McCoy cells, and BGMK cells.

4. The proficiency testing article according to claim 1, characterized in that: When in use, the concentration of the proficiency test item is adjusted by adding different volumes of reconstitution solution to the lyophilized preparation, corresponding to different CT concentrations in clinical samples, covering high, medium and low concentrations.

5. A method for preparing the Chlamydia trachomatis proficiency testing article according to claim 1, comprising the steps of: The CT-sensitive cell line is cultured in a 6-well plate, and after growing into a monolayer of cells, the cell culture medium is removed and a cell culture medium containing cycloheximide is added; CT serotype E is then inoculated, and after completion of the inoculation, the cells are centrifuged and cultured, and the growing CT inclusion bodies are observed. The infected cells are scraped off with a cell brush, ultrasonically disrupted, and centrifuged to remove cell debris, and the CT is then centrifuged to precipitate the CT; finally, the cells are suspended in PBS, inactivated, aliquoted, freeze-dried, and stored at low temperature for later use to obtain a lyophilized powder for Chlamydia trachomatis proficiency testing items; before use, the lyophilized powder is removed, equilibrated to room temperature, and then dissolved in a reconstitution solution.

6. A laboratory application of the Chlamydia trachomatis proficiency testing article according to claim 1.

7. The use according to claim 6, characterized in that: The applications include clinical laboratory external quality assessment, internal quality control and test kit performance evaluation.

8. The use according to claim 6, characterized in that: The application methods include colloidal gold method, latex method and molecular diagnostic technology experimental method based on nucleic acid detection.

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