RU2566071C2 - Method of preparing biomaterial for pcr diagnostics of bovine leukaemia virus (blv) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of veterinary. Method of preparing biomaterial for PCR diagnostics of bovine leukaemia virus (BLV) is claimed. Sampling of animal blood is realised. To prepare for PCR diagnostics samples of biomaterial in form of CC81cell culture, cultivated on Eagle MEM medium in form of multinuclear cell formations - syncytia. As negative control leukocytes from BLV-negative animal, as well as uninfected culture are used.

EFFECT: increased accuracy of BLV by means of PCR is achieved due to peculiarities of cell line of cat pulmonary fibroblasts CC81 to form syncytium in case of presence of virus in samples.

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Description

The invention relates to the field of veterinary medicine and virology, in particular to methods for laboratory diagnosis of the cattle leukemia virus (VL cattle).

Leukemia cattle - a malignant viral lymphoproliferative disease. Retroviridae family VL retrovirus, belonging to the family Retroviridae, genus Deltaretrovirus, integrates into the host leukocyte DNA and remains latent in a large number of cells for a long period of time, which makes it difficult to identify and identify diseased animals.

There are various diagnostic methods for testing cattle blood samples for the presence of cattle VL virus, in particular, serological methods (RID, ELISA) and the polymerase chain reaction (PCR) method. However, using serological methods, it is impossible to detect VL of cattle in the early stages of infection, which is especially important for the timely isolation of healthy calves from VL of cattle of infected animals. PCR is a more sensitive method, but with a low load of the VL overhead cattle provirus in the initial stages of the disease, this method is not always effective (see RF patents No. 2377962; 2379683; 2445370).

A syncytial test is also known that is used to study bovine leukemia virus (Bovine virus) at the Veterinary Research Institute in Pulawy, Poland (Jan Stec, Leokadia Bicka, Bozenna Kozaczynska, Jacek Kuzmak. Lymphoproliferation assay in cattle naturally infected with bovine leukemia (BLV) and bovine immunodeficiency virus (BIV). Bulletin of Veterinary Institute. Pulawy 45, 117-123, 2001). However, this method has several disadvantages. Firstly, with the slightest violation of sterility, contamination of samples is possible, especially if there are several of them in one culture plate. In addition, due to the fact that the culture of CC81 cells is sensitive to several viruses of the Retroviridae family, to confirm the diagnosis of VL cattle infection by the method, it is necessary to carry out a control immunoperoxidase test, which requires additional manipulations, time and costly consumables and solutions (for the total time diagnosis 10-11 days).

The objective of the invention is to provide a method for preparing biomaterial for PCR for the diagnosis of cattle leukemia virus, which allows to determine the presence of VL cattle in blood samples of calves in the early stages of infection and to diagnose using existing serial equipment and test systems and not requiring additional special training and internships for employees diagnostic laboratories with a reduction in the total time of diagnosis, capable of detecting a disease in calves from 15-30 days of age.

The problem is solved due to the characteristics of the CC81 lung cat fibroblast cell line to form syncytium in the presence of VL cattle virus samples, increasing the copy number of the virus by replicating the virion in the studied samples to increase the accuracy of virus detection using PCR, while the method of preparing biomaterial for PCR virus diagnosis bovine leukemia (VL cattle), including taking blood samples of an animal, preparing a sample for research and diagnosing a prepared sample, is characterized by that in order to prepare for PCR diagnostics, biomaterial samples are used in the form of a CC81 cell culture cultured on Eagle MEM medium in the form of multinuclear cell formations - syntyceia, while for the cultivation of CC81 cells, Eagle MEM nutrient medium with L-glutamine and a double set of amino acids with the addition of embryonic calf serum, and the concentration of serum in the nutrient medium is 10%, and for sterilization of serum use heating at a temperature of 56 ° C for 30 minutes, and the culture is cultivated in eraser bottles at a temperature of 37 ° C with a complete change of medium 3 times a week, and when infected with CC81 on Wednesday, a MEM needle with 10% fetal bovine serum and three leukocytes are added with antibiotics: penicillin 100 U / cm ³ , streptomycin 100 μg / cm ³ , amphotericin 5 IU / ml, as well as glutamine, while leukocytes from an animal negative for cattle VL negative, as well as an uninfected culture, are used as a negative control.

The proposed method for preparing biomaterial for PCR for the diagnosis of cattle leukemia virus is based on well-known observations of the Argentinean scientist Ferrer JF, who described the ability of a cell culture to form syncytium under the influence of VL cattle virus (see Ferrer JF, Piper C.E., Abt DA, Marshak RR Diagnosis of bovine leukemia virus infection: evaluation of serologic and hematologic tests by a direct infectivity detection assay. American Journal of Veterinary Research, 1997; Dec. 38 (12): 1977-81).

The developed method for detecting VL of cattle using a CC81 cell culture, unlike the prototype, includes a number of significant changes, which is the novelty of the invention.

Unlike the methodology that served as the prototype for this invention, the subsequent concentration of fetal bovine serum in the medium is reduced to 6% and 2%, instead of 8% and 6% (2 and 4 days after infection, respectively). According to observations, the used serum concentrations have a positive effect on the infection of the cell culture with a virus, presumably contained in the leukocytes of VL cattle of a positive animal.

Also new is that instead of time-consuming and economically disadvantageous peroxidase test, to confirm the presence of VL cattle virus in samples with the formation of syncytium, the method uses an additional refinement test of cell culture on the 7th day after infection by polymerase chain reaction (PCR).

The syncytial test is described in more detail in the following sequence in the above example:

A CC81 cell culture is pre-prepared, 2 ml per well are instilled into a 12-well plate and incubated for 2 days at 37 ° C in a CO ₂ thermostat. After 2 days, a leukocyte suspension obtained from blood samples of the studied cattle is added to the formed CC81 monolayer as follows:

In parallel with the preparation of the CC81 cell culture in animals with suspected leukemia, blood sampling was carried out in vacuum tubes with 10 ml EDTA. The collected blood is centrifuged at 2600 rpm for 30 minutes. Next, you need to remove the supernatant with a pipette and collect the light-colored interphase at the border of plasma and red blood cells containing white blood cells. To this liquid was added 4 ml of sterile distilled cold water, cooled to a temperature of 4-8 ° C, stirred for 30 seconds and 1 ml of sterile 4.5% sodium chloride was added, followed by stirring. Then centrifugation is repeated at 2600 rpm for 30 minutes. The supernatant is removed with a pipette and the precipitate is resuspended in 8 ml of sterile phosphate buffer. Centrifugation is carried out at 1500 rpm for 15 minutes Remove the supernatant and re-wash the precipitate by adding 8 ml of phosphate buffer, shake vigorously. Centrifuge again at 1,500 rpm for 15 minutes. The resulting leukocyte suspension is resuspended in 6 ml of 10% medium with the addition of 3 antibiotics: penicillin, streptomycin (100 units (μg / cm ³ ), amphotericin (5 units / ml) and glutamine. All centrifugation steps are carried out at room temperature. White blood cells from an animal positive for VL cattle are used as a positive control. White blood cells from an animal negative for VL cattle and an uninfected culture are used as a negative control. A leukocyte suspension resuspended in 10% antibiotic medium is digested in lunches of the tablet containing a 2-day monolayer (three wells per sample) (Fig. 1).

The preparation of phosphate buffer (without Ca ⁺⁺ and Mg ⁺⁺ ), which is used to obtain leukocyte suspension, is carried out as follows:

NaCl - 4 g

KCl - 0.1 g

Na ₂ HPO ₄ × 12H ₂ O - 1.45 g

KN ₂ RO ₄ - 0.1 g

Redistillate water - up to 500 ml

Counting the number of leukocytes is carried out in the Goryaev’s chamber. To infect the CC81 monolayer, at least 5 × 10 ⁶ cells in 1 ml of medium must be present.

2 and 4 days after infection of the culture medium from the wells is removed and fresh medium is added with a serum concentration of 6% and 2%, respectively. In FIG. 2A depicts a CC81 monolayer 2 days after adding a nutrient medium containing leukocytes from VL cattle positive ram. On the 7th day after infection of the culture, the cells are washed three times with phosphate buffer and stained according to the Grunwald-Giemsa method as follows:

Grunwald's solution is added for 3 minutes, while the wells should be completely covered with liquid. Next, the Grunwald solution is carefully removed with a dispenser and the cells are washed several times with distilled water. Then, a fresh Giemsa solution is added for 15 minutes (1.5 ml of solution and 23.5 ml of distilled water are prepared). After that, the tablet is carefully washed with water. The stained cell culture is examined using a microscope. CPD caused by VL of cattle is observed in the form of syncytia - multinuclear cell formations (Fig. 2, C, D). A monolayer of intact CC81 cell culture as a negative control is depicted in FIG. 2B. Next, the resulting cell culture is sent for PCR diagnosis.

In FIG. 1. A schematic representation of a 12-well culture plate containing a 2-day CC81 monolayer after adding 10% medium MEM needle (see above) in the form of 12 pink circles is shown. Small yellow circles represent white blood cells. Wells A1, B1, C1: a leukocyte suspension from a negative animal by VLCRS (negative control) was added on Wednesday; wells A2, B2, C2: a leukocyte suspension from the test animal was added to the medium; wells A3, B3, C3: a leukocyte suspension from an animal positive for VL cattle was added on Wednesday (positive control); wells A4, B4, C4: uncharged monolayer as an additional negative control.

In FIG. Figure 2 shows images of a CC81 cell culture obtained with an inverted Carl Zeiss Axio Observer A1 microscope. A: monolayer of cell culture CC81 2 days after adding 10% of the medium (see above) containing leukocytes obtained from sheep blood positive for VL cattle. White blood cells are marked by arrows; B: monolayer of uninfected CC81 culture on the 7th day of the syncytial test. B, D: examples of the formation of syncytia in a monolayer of CC81 cell culture on the 7th day after leukocyte infection from VL cattle of a positive ram. The arrows indicate the formation of syncytia containing 4 nuclei (B) and 5 nuclei (G), respectively. Cell culture was stained according to the method of Grunwald-Giemsa. The nuclei and cytoplasm are pink and purple, respectively.

The experiments to refine the parameters of the proposed method were carried out on the basis of the department for monitoring and predicting infectious animal diseases of the Federal State Budget Scientific Institution "Ural Scientific Research Veterinary Institute" in Yekaterinburg. The proposed method has been tested with positive results and regular reproducibility on samples and blood samples obtained from dysfunctional farms of the Sverdlovsk, Kurgan and Tyumen regions of the Urals Federal District, including the diagnosis of blood samples of calves aged 15 days to 3 months, which by other diagnostic methods previously almost impossible to do.

Secondary signs of the method, such as the use of penicillin, streptomycin (100 PIECES (μg / cm ³ )), amphotericin (5 PIECES / ml) and glutamine, the concentration of fetal serum in the nutrient medium 10%, as well as others, as an additive along with leukocytes signs, identified and justified almost empirically and provide the stated end result.

Implementation of the proposed method, which can be considered an early diagnosis of the causative agent of leukemia based on PCR, will determine the virus carriers in young cattle from the age of 15-20 days.

The developed early diagnosis of leukemia will increase the efficiency of recovery of agricultural enterprises from infection.

An unobvious effect of the proposed method is that due to the preparation of biomaterial for PCR diagnostics according to the proposed scheme with growing a culture of CC81 cells on Igla MEM medium at specified parameters, the copy number of the virus increases, which increases the efficiency of PCR diagnostics and allows detection of virus carriers among calves of embryonic infection at the age of 15 -30 days, reducing the cost of improving the herd.

Claims (1)

A method for preparing biomaterial for PCR for diagnosis of cattle leukemia virus (VL cattle), including taking blood samples of an animal, preparing a sample for research and diagnosing a prepared sample, characterized in that for preparation for PCR diagnosis using biomaterial in the form of a cell culture CC81 cultured on the medium Needle MEM in the form of multinuclear cell formations - syntyntia, while for the cultivation of CC81 cells using nutrient medium Needle MEM with L-glutamine and a double set mineral acids with the addition of fetal calf serum, and the serum concentration in the nutrient medium is 10%, and for sterilization of the serum, heating is used at 56 ° C for 30 minutes, and the culture is cultivated in plastic bottles at 37 ° C with a complete change of medium 3 times per week, and when infected with CC81 on Wednesday, a MEM Eagle with 10% fetal bovine serum and three leukocytes are added with antibiotics: penicillin 100 IU / cm ³ , streptomycin 100 μg / cm ³ , amphotericin 5 IU / ml, and glutamine, as neg -negative control leukocytes from the animal is used, negative for VL RNC and uninfected culture.

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