CN-121978351-A - Method for quantitatively detecting viable count of mycoplasma bovis

Abstract

The invention belongs to the technical field of quantitative detection of microorganisms, and particularly relates to a method for quantitatively detecting the number of live mycoplasma bovis. According to the method, after various experimental conditions such as bacterial liquid preservation conditions, ultrasonic crushing modes, crushing time, sample processing modes and the like are optimized, the linear relation between the protein concentration and the viable count of the multi-batch mycoplasma bovis liquid samples is explored, and finally the quantitative detection method for the viable count of mycoplasma bovis is obtained.

Inventors

• HE SUN
• SONG JIANGXIA
• FENG HUIYUN
• HOU YUZHEN
• HAO CHENGWU
• PAN XIAOMEI
• LING CHEN
• ZHANG FEI
• HAN RUI
• CAO JIAN
• HU YAOKAI

Assignees

• 天康生物制药有限公司

Dates

Publication Date

20260505

Application Date

20260228

Claims (10)

1. 1. The method for quantitatively detecting the viable count of mycoplasma bovis is characterized by comprising the following steps: and (3) carrying out centrifugal resuspension on the mycoplasma bovis bacterial liquid to obtain a resuspension bacterial liquid, detecting the protein concentration, and calculating according to a fitting standard curve of the protein concentration and the mycoplasma bovis viable count to obtain the mycoplasma bovis viable count.
2. 2. The method of claim 1, wherein the mycoplasma bovis solution is a solution that is stored at 2-8 ℃ for 0-24 hours.
3. 3. The method according to claim 1, wherein the protein concentration of the resuspended bacterial liquid is detected by any one of the following methods (1) or (2): (1) Directly detecting the protein concentration of the re-suspension bacteria liquid; (2) Crushing the re-suspended bacteria liquid, centrifuging again, and taking supernatant to detect the protein concentration.
4. 4. A method according to claim 3, wherein the disruption is non-contact energy-accumulating ultrasonic disruption.
5. 5. The method of claim 4, wherein the non-contact energy-gathering ultrasonic crushing is performed for 50-60 min.
6. 6. A method according to claim 3, wherein the re-centrifugation is performed for 10-20min at 10000-12000 r/min.
7. 7. The method of claim 3, wherein when the protein concentration is obtained by directly detecting the resuspended bacteria liquid, the fitted standard curve is as shown in formula I: y= 0.0106*x-0.4478 (I), Wherein x is the protein concentration obtained by direct detection, mug/mL, y is the viable count, and 10 9 CFU/mL.
8. 8. The method of claim 3, wherein when the protein concentration is obtained by crushing the resuspended bacteria liquid and then centrifuging again to obtain supernatant, the fitted standard curve is shown in formula II: y=0.111*x1-0.2967 (II), Wherein x1 is the protein concentration obtained by crushing and then centrifuging again to obtain supernatant for detection, mug/mL, and y is the viable count of 10 9 CFU/mL.
9. 9. The method of claim 1, wherein the mycoplasma bovis bacteria solution comprises a bacteria solution obtained by fermentation culture of mycoplasma bovis.
10. 10. The method of claim 1, wherein the resuspension is performed using PBS buffer.

Description

Method for quantitatively detecting viable count of mycoplasma bovis Technical Field The invention belongs to the technical field of quantitative detection of microorganisms, and particularly relates to a method for quantitatively detecting the number of live mycoplasma bovis. Background The existing mycoplasma bovis viable bacteria counting adopts a plate counting method, and the method adopts an experimental principle that samples are diluted and then coated on a solid culture medium, and each viable bacteria grows to form a visible colony (CFU, colony forming unit). The specific method of the experiment is that the samples are serially diluted (usually 10-time gradient dilution), a proper amount of diluent is taken for coating or pouring the flat plate, and colonies are counted after culturing under proper conditions. The method has the advantages of directly reflecting the number of living bacteria, but also has the disadvantages that the time is long (24-48 hours), the counting result is influenced by personnel counting errors, and only the microorganisms which can be cultured can be counted. Disclosure of Invention The invention aims to solve the technical problem of providing a method for quantitatively detecting the number of live mycoplasma bovis by aiming at the defects of the prior art. In order to solve the technical problems, the invention discloses a method for quantitatively detecting the number of live mycoplasma bovis. The specific technical scheme is as follows: the invention provides a method for quantitatively detecting the number of live mycoplasma bovis, which comprises the following steps: And (3) carrying out centrifugal resuspension on the mycoplasma bovis bacterial liquid to obtain a resuspension bacterial liquid, detecting the protein concentration, and calculating according to a fitting standard curve of the protein concentration and the mycoplasma bovis viable count to obtain the mycoplasma bovis viable count. The method is a non-disease diagnostic method. Wherein the mycoplasma bovis bacterial liquid is preserved for 0-24 hours at the temperature of 2-8 ℃. Wherein the protein concentration of the resuspended bacteria liquid is detected according to any one of the following methods (1) or (2): (1) Directly detecting the protein concentration of the re-suspension bacteria liquid; (2) Crushing the re-suspended bacteria liquid, centrifuging again, and taking supernatant to detect the protein concentration. Wherein the crushing is non-contact energy-gathering ultrasonic crushing. The non-contact energy-gathering ultrasonic crushing is carried out for 50-60 min. In some embodiments of the invention, the ultrasonic crushing procedure is ultrasonic for 15-25 s and stopping for 10-15 s. And the secondary centrifugation is performed for 10-20 min at 10000-12000 r/min. In some embodiments of the invention, the re-centrifugation is 12000 r/min centrifugation 10 min. Wherein the protein concentration is detected by using a BCA protein quantitative detection kit. When the protein concentration is obtained by directly detecting the resuspended bacteria liquid, the fitting standard curve is shown as a formula I: y= 0.0106*x-0.4478 (I), Wherein x is the protein concentration obtained by direct detection, mug/mL, y is the viable count, and 10 9 CFU/mL. When the protein concentration is obtained by crushing the re-suspended bacteria liquid and then centrifuging again to obtain supernatant, the fitting standard curve is shown in the formula II: y=0.111*x1-0.2967 (II), Wherein x1 is the protein concentration obtained by crushing and then centrifuging again to obtain supernatant for detection, mug/mL, and y is the viable count of 10 9 CFU/mL. If the protein concentration of the resuspended bacterial liquid prepared from the mycoplasma bovis bacterial liquid is detected after concentration or dilution treatment, the fitting standard curve is obtained by multiplying the dilution factor (or the inverse of the concentration factor) by the formula I or the formula II, specifically: When the protein concentration is obtained by directly detecting the concentrated or diluted re-suspended bacteria liquid, the fitting standard curve is shown as a formula III: y = (0.0106*x - 0.4478)*n (III), when the protein concentration is obtained by crushing the concentrated or diluted re-suspended bacteria liquid, centrifuging again and taking the supernatant for detection, the fitting standard curve is shown as a formula IV: y=(0.111*x1-0.2967)*n (IV)。 n in formulas III and IV is the inverse of the dilution or concentration, for example, 4 times n is 0.25, 4 times n is 4, i.e. the original concentration is reduced. The fitting standard curve of the protein concentration and the mycoplasma bovis viable count is obtained according to the following method that when mycoplasma bovis bacterial liquid is resuspended after centrifugation, concentration or dilution treatment with different multiples is completed by a method