Bacteriophage reagents for the diagnosis of anthrax.
1.Product Specifications:
1ml as |(browser search): CDC Research Reagents Blog
Joint|Department: i99l--47-47--i94
2.Performance metrics.
2.1 Appearance.
It is a yellowish and clear liquid.
2.2 Specificity.
Use more than 3 strains of anthrax strains and 1 strain of Bacillus cereus at the same time for lysis test, only lysage.
Those who dissolve anthrax are qualified.
2.3 Potency determination.
The phage stock solution to be tested was serially diluted 10 times with beef digestive juice broth, and 1 straw should be replaced for each dilution. Take another 3 test tubes, and add 1ml of anthrax (pno.) to each2 strains) juvenile cultures (containing about 30 10 °), then add 1 ml of 10 ° dilution of bacteriophage, shake well, and leave in a 45 water bath for 5 min, immediately on each large.
Add 10g of 10g l agar of the same temperature to the tube, mix well and evenly, be careful not to foam, and pour them separately.
3 flat dishes. Set 35 37 culture for 12 24 h and count the plaques. Find the titer per 1 ml phage stock solution.
pfu/ml) 。The potency should not be less than 10 pfu ml.
3.Test Method.
3.1 Appearance.
Using the visual inspection method, the visual inspection in the bright place of natural light should be in accordance with 21.
3.2 How to use.
Inoculate the fresh juvenile culture of the bacteria to be tested on the surface of the agar plate, add 1 drop of the bacteriophage after 10 minutes, tilt the plate to make the flow into a band, and use standard anthrax as a control. Set 35 37 culture 8 10
hours, the presence of a significant lytic zone is positive.
Appendix A Requirements for Main Raw Materials, Production Processes and Semi-finished Products.
1 Main raw material requirements.
1.1 Bacteriophage.
1.1.1 Bacteriophage species and strains are distributed or approved by the National Drug Control Agency.
1.1.2 Cultures preservation, passage and characteristics.
The bacteriophage species were strains of Anthracnose Phage AP631 (or other superior bacteriophage species). Before manufacturing, the seed stock solution should be counted to determine its potency, which should not be less than 10. pfu/ml 。The strain is attenuated anthrax strain.
pno.2 strains (or other susceptible strains).Strain characteristics should be typical.
2 Main production process requirements.
Anthrax pno2. Strain opening and verification.
Anthrax pno2 Bacterial liquid culture ".
Anthrax bacteriophage AP631
Lysate. Sterilization filtration.
Wrap. Semi-finished product verification.
Finished product verification. Note: 1"* is a key process;"*Special process.
2.Control content and control standards: The microbiological examination is confirmed, and the titer of the seed stock solution should not be less than 10 pfu ml.
3.Special process control content.
The process should be carried out in the biological safety cabinet in the positive operation area, and the personnel should strictly follow the management of the positive operation area.
system and standard operating procedures.
2.1. Preparation of phage and host bacterial solution.
2.1.1 medium.
The broth of beef digestive juice used for manufacturing has an amino nitrogen content of lmg ml and a pH value of 74~7.6。
2.1.2 Inoculation culture.
The 14-16 h fresh culture of the host bacteria was collected in sterilized physiological sodium chloride solution, shaken well, and sampled. The concentration was determined according to the "Chinese Bacterial Corrosion Standard". Take 1 ml of the host bacteria stock solution (containing bacteria 3.)0 10 °) was added to 100ml of beef digest broth, and at the same time inoculated with 1 ml of phage seed stock solution, set 35 37 culture 8 10
hours, and the liquid can be collected when it is transparent.
2.1.3 Sterilization and filtration.
After the collected phage culture medium is sterilized and filtered, it is a semi-finished product.
2.2. Semi-finished product verification.
The verification shall be carried out in accordance with item 3, and shall comply with the regulations.
2.3 aliquots.
Those who pass the sterility test and have a titer of not less than 10 pfu ml will be dispensed. The aliquot size is 1 ml bottle.
2.4. Verification of finished products.
According to the performance indicators and test methods specified in this technical requirement, the results should be in line with 21～2.3 items.
requirements. 3 Semi-finished product requirements.
3.1 Sterility test.
According to the sterility inspection method (Appendix xia) in the Pharmacopoeia of the People's Republic of China (2005 Edition, Part III).
carried out, should be in accordance with the regulations.
3.2 Potency determination.
The phage stock solution to be tested was serially diluted 10 times with beef digestive juice broth, and 1 straw should be replaced for each dilution. Take another 3 test tubes and add 1 ml of Anthracnose (PNO.) to each2 strains) juvenile cultures (containing about 30 10%), then add 1ml of 10 ° dilution of phage, shake well, put in a 45 water bath for 5 minutes, immediately add 10ml of 10g l agar of the same temperature to each large tube, mix well, pay attention not to foam, and pour 3 flat dishes respectively. Set 35 37 culture for 12 24 h and count the plaques. Find.
Potency per 1 ml phage stock solution (pfu ml).
Instructions for bacteriophages for the diagnosis of anthrax.
Product Name] Generic Name: Anthracnose Diagnostic Bacteriophage.
Packing specifications] 1ml
Intended Use] This product is clinically used to identify anthrax.
Test principle] The host bacteria were lysed by phages, and obvious lysis zones appeared for detection.
Main Components] Anthracnose bacteriophage orchid AP631 strain.
Storage conditions and expiration date].
2 8 Store in the dark, valid for 2 years.
The date of manufacture and expiration date are available on the kit packaging label.
Sample Requirements] Fresh juvenile bacterial cultures.
Test method] Inoculate the fresh young culture of the bacteria to be tested on the surface of the agar plate, add 1 drop of the bacteriophage after 10 minutes, and tilt the plate to make the flow into a band. At the same time, standard anthrax bacteria were used as a control. After 35 37 incubation for 8 10 hours, those with obvious lysis zones are positive.
Interpretation of test results] Positive: indicates that the culture is anthracnose.
Limitations of the test method] This test method requires a bacterial isolation culture.
Product performance indicators].
Specificity. With more than 3 strains of anthrax and 1 strain of Bacillus cereus at the same time, only anthrax can be lysed. Potency determination.
The phage stock solution to be tested was serially diluted 10 times with beef digestive juice broth, and 1 straw should be replaced for each dilution. Take another 3 test tubes, and add 1ml of anthrax (pno.) to each2 strains) juvenile cultures (containing about 30 10), then add 10° dilution of phage IML to each of them, shake well, put in a 45 water bath for 5 minutes, and immediately add the same temperature to each large tube.
10 g l of 10 ml of agar, mix well and be careful not to foam, pour 3 flat dishes respectively. Set 35 37 culture for 12 24 h and count the plaques. Find the titer (pfu ml) of each IML phage stock solution. The titer is not less than 10 pfu ml.
Precautions] 1This product is intended for in vitro diagnostic use only.
2.Use the product in strict accordance with this instruction manual, and it is forbidden to freeze.
References] 1Guidelines for the preparation of instructions for in vitro diagnostic reagents.
2.Regulations of Chinese Biological Products, 2000 edition.