African green monkey kidney cell clone and its application in detection of newcastle disease virus of chicken
By using the African green monkey kidney cell clone VERO-K6, the detection process for Newcastle disease virus in chickens has been simplified, costs have been reduced, and the detection cycle has been shortened, achieving efficient detection of virus content.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ANHUI DIVINITY BIOLOGICAL PROD CO LTD
- Filing Date
- 2023-04-12
- Publication Date
- 2026-05-29
AI Technical Summary
The existing Newcastle disease virus testing process for chickens is cumbersome, has a long testing cycle, and is costly.
The African green monkey kidney cell clone VERO-K6 was used as the host cell. By diluting the virus sample and inoculating it into cell culture plates, cytopathic effects were observed, and TCID50 was calculated using the Reed-Muench method, which simplified the detection process and shortened the detection cycle.
The testing cost has been reduced, the process has been simplified, the testing cycle has been shortened from 15 days to 6 days, and the accuracy of the results is similar to that of the chicken embryo method.
Smart Images
Figure CN116286606B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biological product testing technology, specifically to a cloned strain of African green monkey kidney cells and its application in the detection of Newcastle disease virus in chickens. Background Technology
[0002] In the field of biological product testing, virus content detection is a very important process. In order to obtain a better detection method, the selection of the host for virus culture is also crucial. Different viruses correspond to different hosts, while the same virus can infect multiple hosts. Using the optimal host to detect the virus will result in high accuracy.
[0003] According to the 2000 edition of the "Regulations for Veterinary Biological Products of the People's Republic of China" and the 2020 edition of the "Veterinary Pharmacopoeia of the People's Republic of China" (Volume 3), the chicken embryo method is used to test the content and efficacy of Newcastle disease virus in chickens.
[0004] The original detection method mainly involved serially diluting Newcastle disease virus (NDV) solution 10-fold with sterile physiological saline. Three suitable dilutions were obtained, and 0.1 ml of the solution was inoculated into the allantoic cavity of five 10-day-old SPF chicken embryos, one at a time. The embryos were incubated at 37°C for 120 hours. Embryos that died within 48 hours were discarded. Embryos that died between 48 and 120 hours were removed, and their allantoic fluid was harvested. Equal volumes of allantoic fluid at the same dilution were mixed, and the hemagglutination titer was determined. After 120 hours, all viable embryos were removed, and their allantoic fluid was harvested individually for further hemagglutination titer determination. Infection was defined as an allantoic fluid titer of at least 1:160 (1:128 using the micro-method). The EID was calculated using the Reed-Muench method. 50 This chicken embryo testing method uses SPF chicken embryos, which have high raw material costs; after inoculation, the eggs need to be candled regularly, which increases labor costs; when determining the results, the air cell of the chicken embryo needs to be knocked off the eggshell and allantoic fluid needs to be collected to measure the hemagglutination titer, making the testing process cumbersome; and the testing cycle is long, taking 15 days from the start of incubation to the end of testing.
[0005] Chinese patent document CN106435021A discloses a kit for detecting different genotypes of Newcastle disease virus (NDV) in chickens. This invention provides a kit for detecting different genotypes of NDV and belongs to the field of RT-PCR detection technology. The invention also provides a method for detecting different genotypes of NDV. The kit and detection method of this invention are characterized by high specificity, high sensitivity, high efficiency, good versatility, and low cost. They can rapidly differentiate and diagnose clinical samples within 6 hours. This invention provides technical means for the early rapid diagnosis of NDV and the conduct of molecular epidemiological investigations, thereby better guiding the prevention and control of this disease in poultry production. However, this patent can only detect different genotypes of NDV and cannot rapidly detect the NDV content. Summary of the Invention
[0006] The technical problem to be solved by this invention is how to solve the problems of cumbersome detection process, long detection cycle and high cost of existing Newcastle disease virus detection in chickens.
[0007] The present invention solves the above-mentioned technical problems through the following technical means:
[0008] Currently, almost all viruses can find corresponding cell hosts. My team screened dozens of cell types and explored various detection processes. After three years, we finally selected a VERO-K6 cell strain that is sensitive to Newcastle disease virus in chickens. The detection results are basically consistent with those obtained by the chicken embryo method.
[0009] The first aspect of this invention provides an African green monkey kidney cell clone, VERO-K6, which has been deposited at the China Center for Type Culture Collection (Wuhan University), accession number CCTCC NO: C2022312, deposit date: March 2, 2023.
[0010] A second aspect of the present invention provides an application of the above-mentioned African green monkey kidney cell clone VERO-K6 in the detection of Newcastle disease virus in chickens.
[0011] A third aspect of the present invention provides a method for detecting the content of Newcastle disease virus in chickens using the above-mentioned African green monkey kidney cell clone VERO-K6, comprising the following steps:
[0012] (1) Cell plating:
[0013] Take out the VERO-K6 cell suspension, put a certain amount into a petri dish, and then add it into the well plate of the cell culture plate using a pipette. Place the cell culture plate in an incubator and culture until it grows into a dense monolayer of cells.
[0014] (2) Sample dilution:
[0015] Take a certain number of EP tubes and place them on a centrifuge rack. Label the EP tubes in order and add an equal amount of DMEM serum-free diluent containing trypsin to each EP tube. Take a certain amount of chicken Newcastle disease virus solution and add it to the first EP tube. Shake and mix well. Take an equal amount of chicken Newcastle disease virus solution from the EP tube and add it to the second EP tube. Shake and mix well. Repeat this process for the remaining EP tubes.
[0016] (3) Sample addition:
[0017] Remove the cell culture plate from step (1) where dense monolayers of VERO-K6 cells have grown, discard the cell culture medium, and wash with PBS solution; take the diluted chicken Newcastle disease virus solution from the EP tube in step (2) and inoculate it into the cell culture plate;
[0018] (4) Culture and detection:
[0019] Place the cell culture plate in an incubator and incubate. Record the number of effective lesion wells on the cell culture plate and calculate TCID using the Reed-Muench method. 50 .
[0020] Preferably, the density of the VERO-K6 cell suspension in step (1) is 300,000 to 500,000 cells / mL.
[0021] Preferably, the cell culture plate in step (1) is a 96-well cell culture plate.
[0022] Preferably, the amount added to the orifice plate in step (1) is 100 μL / well.
[0023] Preferably, the temperature inside the incubator in step (1) is 37°C and the incubation time is 24 hours.
[0024] Preferably, the DMEM serum-free diluent in step (2) contains 5-15 μg / mL of pancreatic enzyme.
[0025] Preferably, in step (2), the volume ratio of DMEM serum-free diluent containing trypsin to a certain amount of chicken Newcastle disease virus solution is 9:1.
[0026] Preferably, in step (4), the incubator contains 5% CO2 and the temperature inside the incubator is 37°C.
[0027] The advantages of this invention are:
[0028] 1. Compared to the chicken embryo method, the cost of raw materials and auxiliary materials is lower.
[0029] 2. The testing process is simple, requiring only observation after inoculation. For the chicken embryo method, eggs are candled every 6 hours after inoculation for 48 hours, requiring night shifts. The hemagglutination titer needs to be measured 120 hours after inoculation.
[0030] 3. The testing cycle is short, taking only 6 days from cell plating to the end of the testing process. The chicken embryo method takes 15 days from hatching the chicken embryos to obtaining the results.
[0031] 4. Compared with existing methods for detecting Newcastle disease virus in chickens, this method has lower testing costs, a simpler and easier testing process, and a shorter testing cycle. Attached Figure Description
[0032] Figure 1 This is a schematic diagram of the detection mark of the present invention;
[0033] Figure 2 This is a sample gradient dilution diagram of the present invention;
[0034] Figure 3 A diagram of healthy cells;
[0035] Figure 4 This is a diagram of the diseased cells. Detailed Implementation
[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0037] Sample 1: Newcastle disease antigen for chickens, produced by Anhui Dongfang Diwei Biological Products Co., Ltd.
[0038] Sample 2: Newcastle disease live vaccine for chickens, produced by Anhui Dongfang Diwei Biological Products Co., Ltd.;
[0039] Sample 3: Newcastle disease live vaccine for chickens, produced by Liaoning Yikang Biotechnology Co., Ltd.
[0040] Example 1:
[0041] Cell screening methods:
[0042] I. Screening of Sensitive Cell Lines:
[0043] Cell lines: three ST cell lines from different sources (WH-ST, SZ-ST, SH-ST), five BHK21 cell lines from different sources (WH-BHK21, NJ-BHK21, SH-BHK21, JH-BHK21, BJ-BHK21), four Vero cell lines from different sources (WH-Vero, SZ-Vero, SH-Vero, BJ-Vero), three Marc145 cell lines from different sources (SH-Marc145, JN-Marc145, JH-Marc145), and four PK-15 cell lines from different sources (FZ-PK-15, BJ-PK-15, GS-PK-15, SH-PK-15), which were numbered respectively.
[0044] 1. Inoculate each of the above cell lines with the same amount of Newcastle disease virus solution, and culture continuously for 120 hours using DMEM maintenance medium containing 2% newborn calf serum to observe cytopathic effects.
[0045] 2. Inoculate each of the above cell lines with the same viral load of Newcastle disease virus solution, and culture continuously for 120 hours using DMEM maintenance medium containing trypsin but not newborn calf serum to observe cytopathic effects.
[0046] Results: SZ-Vero cells were found to show obvious and typical lesions in DMEM maintenance medium containing trypsin but not newborn calf serum. From this, the cells with the highest sensitivity to Newcastle disease virus were screened out from 19 cell lines and labeled as Vero-L.
[0047] II. Single-cell cloning:
[0048] Vigorous Vero-L cells that formed a well-developed monolayer were selected, digested, and then serially diluted in 96-well plates using a limiting dilution method. The diluted cells were seeded into 96-well plates and cultured at 37°C in a 5% CO2 incubator. Subcloning was performed on selected single-cell clones, resulting in three cell lines: VERO-K6, VERO-K8, and VERO-K9. The selected single-cell clones were then expanded by seeding each well with 200 μL (containing 30,000–50,000 cells) into 96-well plates in quadruplicates. Control wells were also included. Cell growth rate and status were observed at 24, 48, 72, and 96 hours. The results showed that the growth rate of the three cell lines was: VERO-K9 ≥ VERO-K6 > VERO-K8.
[0049] The susceptibility of three clonal cell lines to Newcastle disease virus (NDV) was tested: Cloned VERO-K6, VERO-K8, and VERO-K9 cells were passaged using normal methods, resulting in dense monolayers. These cells were then inoculated with NDV and a maintenance medium containing trypsin but no newborn calf serum (NCS). Cytopathic effects were observed from 24 hours to 120 hours. VERO-K6 cells showed significant cytopathic effects, followed by VERO-K9 cells, while VERO-K8 cells showed only minor cytopathic effects. This completed the screening of VERO-K6 cells as NDV-sensitive clones.
[0050] The VERO-K6 cells have been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2022312 and deposit date: March 2, 2023.
[0051] Example 2:
[0052] A method for detecting Newcastle disease virus content in chickens using the above-mentioned VERO-K6 includes the following steps:
[0053] (1) Cell plating:
[0054] Take out a VERO-K6 cell suspension with a density of 300,000 to 500,000 cells / mL and add it to the wells of a 96-well cell culture plate using a pipette, 100 μL / well. Fix the 96-well cell culture plate with masking tape and then place it in a carbon dioxide incubator (5% CO2, 37°C, 24h) to grow into a dense monolayer of cells.
[0055] (2) Sample dilution:
[0056] Place nine 2.0 mL EP tubes on a centrifuge rack and label them sequentially (10...). -1 10 -2 10 -3 ...10 -9 Add 900 μL of DMEM serum-free diluent containing 10 μg / mL trypsin to each EP tube; add 100 μL of chicken Newcastle disease virus solution (sample 1, Dongfang Diwei chicken Newcastle disease antigen) to a tube labeled 10. -1 The mixture was vibrated and mixed in the EP tube, from which the 10 -1 Draw 100 μL of liquid from the EP tube and add it to 10 -2 The mixture is vibrated and mixed in the EP tube, and so on, the same operation is performed on the remaining EP tubes (e.g. Figure 2 (as shown);
[0057] (3) Sample addition:
[0058] Remove the 96-well cell culture plate from step (1) where a dense monolayer of VERO-K6 cells has formed, discard the cell culture medium, and wash twice with PBS solution; from step (2) 10 -6 10 -7 10 -8 Take an equal amount of chicken Newcastle disease virus solution from each of the three dilutions and inoculate it into a cell monolayer, with 8 wells per dilution (100 μL / well);
[0059] Instructions: In the negative control wells, add 8 wells of DMEM serum-free dilution buffer containing 10 μg / ml trypsin to form a cell monolayer (100 μL / well); in the positive control wells, add 8 wells of DMEM serum-free dilution buffer containing 10 μg / ml trypsin to form a cell monolayer. -1 Virus solution was poured into a cell monolayer as a positive control. The cells were labeled (sample number, dilution, and test date), as shown in the labeling instructions. Figure 1 As shown.
[0060] (4) Culture and detection:
[0061] The 96-well cell culture plates were fixed with masking tape and then placed in an incubator (37°C, 5% CO2) for 5 days. The number of effective lesion wells on the cell culture plates was recorded, and the TCID was calculated according to the Reed-Muench method. 50 .
[0062] Example 3:
[0063] The difference between this embodiment and Embodiment 2 is that: in step (2), "Newcastle disease virus solution (sample 1, Oriental Diwei's Newcastle disease antigen)" is changed to "Newcastle disease virus solution (sample 2, Oriental Diwei's live Newcastle disease vaccine, diluted to 1 dose / 0.1ml)", and in step (3), "10 -6 10 -7 10 -8 "Three dilutions" changed to "10" -4 10 -5 10 -6 "Three dilutions", the other steps are the same as in Example 2.
[0064] Example 4:
[0065] The difference between this embodiment and embodiment 3 is that the "Newcastle disease virus liquid (sample 2 Oriental Diwei's Newcastle disease live vaccine, diluted to 1 dose / 0.1ml)" in step (2) is changed to "Newcastle disease virus liquid (sample 3 Liaoning Yikang's Newcastle disease live vaccine, diluted to 1 dose / 0.1ml)", and the other steps are the same as in embodiment 3.
[0066] The chicken embryo method was then used to test samples 1-3. The results of the two detection methods (chicken embryo method and cell method, i.e., Examples 2-4) are compared as follows:
[0067] Detection methods Sample 1 Sample 2 Sample 3 <![CDATA[Chicken embryo method (EID 50 )]]> <![CDATA[10 8.2 EID 50 / 0.1ml]]> <![CDATA[10 6.5 EID 50 / feather]]> <![CDATA[10 6.4 EID 50 / feather]]> <![CDATA[Cell method (TCID 50 )]]> <![CDATA[10 9.2 TCID 50 / ml]]> <![CDATA[10 7.6 TCID 50 / ml]]> <![CDATA[10 7.3 TCID 50 / ml]]>
[0068] Convert the chicken embryo method test results to per milliliter, sample 1:10 9.2 EID 50 / ml, Sample 2:10 7.5 EID 50 / ml, Sample 3:10 7.4 EID 50 / ml. The results of the two methods are basically consistent with those of the cell method, which shows that the cell method can replace the chicken embryo method for the detection of Newcastle disease virus in chickens.
[0069] Figure 1 This is a schematic diagram of the detection mark of the present invention; Figure 2 This is a sample gradient dilution diagram of the present invention; Figure 3 A diagram of healthy cells; Figure 4 This is a diagram of the diseased cells.
[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A clone of African green monkey kidney cells, VERO-K6, characterized in that, The clone VERO-K6 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2022312 and deposit date: March 2, 2023.