Genetic Fingerprinting of Bacillus thuringiensis Isolates by Randomly Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR)

Genetic Fingerprinting of Bacillus thuringiensis Isolates by Randomly Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR)

(Published in Nepal Journal of Science and Technology 10(2009) 97-103)

Gyan Sundar Sahukhal^l, Upendra Thapa Shrestha¹, Binod Lekhak², Anjana Singh², Viswanath Prasad Agrawal¹

¹Research Laboratory for Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal.

²Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal

e-mail: gyan633413@gmail.com

Abstract

Random Amplified Polymorphic DNA (RAPD) is a method of producing a genetic fingerprint of a particular species without its prior genetic information. Relationship between species may be determined by comparing their unique fingerprint information. B. thuringiensis was isolated from soil samples of Khumbu base camp of Everest region, Nepal. Crystal protein (delta endotoxin) producing strains (46 from Phereche and 40 from Sagarmatha national park) were tested against a series of 100 decamer RAPD primers (codes 201-300, obtained from University of British Columbia) by RAPD PCR. Primer 284 was found the best among the tested primers and the reaction condition for PCR was optimized with a PCR buffer containing 10mM Tris HCl, 50 mM KCl, 3 mM MgCl₂ with pH 8.3.; 200μm dNTPs each, 1U Taq polymerase , 40 pmol decamer primers, 20 ng template DNA and 1% DMSO as a final concentrations in 25μl reaction mixture. The thermal programme was programmed as initial denaturation temperature at 94^oC for 5 min followed by 35 cycles with denaturation at 94^oC for 1 min, annealing at 36^oC for 1 min and extension at 72^oC for 2 mins with final extension temperature at 72^oC for 10 min. Higher polymorphic fragments were found in the range between 700-900 bp. Next to it, the range of 400-700 and 1200-1600 bp were, too, highly polymorphic among the isolates. The discriminatory capacity (D) of the RAPD-PCR was found to be 0.9901. The isolates of cold tolerant B. thuringiensis from high altitude regions were found rich in genomic polymorphism.

Key words: RAPD-PCR, fingerprint, endotoxin, Bacillus thuringiensis Berliner, polymorphism, base pair (bp)

Viral Diseases and its control measure

List of Epidemic Viral Diseases of Man
Following is the list of such important disease of man that appears in epidemic form:

Disease	Pathogen	Infection Sources	Reservoirs	Control Measures
German Measles	Rubella virus	Human cases	Humans	Immunization; avoid contact between infected individuals and pregnant women
Influenza	Influenza virus	Human cases	Humans, animals	Immunization
Measles	Measles virus	Human cases	humans	Immunization
Acquired immuno deficiency syndrome (AIDS)	Human immuno-deficiency virus (HIV)	Infected body fluids, especially blood and semen	Humans	Treatment with reverse transcriptase inhibitors, protease inhibitors (not curative)
Rabies	Rabies virus	Bite by carnivores	Wild and domestic carnivores	Avoid animal bites; immunization of animals handlers and exposed individuals
Hantavirus pulmonary syndrome	Hantavirus	Inhalation of contaminated fecal matter; contact	Rodents	Control of rodent population and exposure
Hemorrhagic fever	Ebola virus	Infected body fluids	Unknown fluids	Quarantine of active cases
Hepatitis	Hepatitis A,B,C,D,E	Infected humans	Humans	Decontamination of contaminated fluids and formites, immunization if available

Bacterial Diseases and its control measure

List of Epidemic Bacterial Diseases of Man

Following is the list of important epidemic disease of man caused by bacterial pathogens:

Disease	Pathogen	Infection Sources	Reservoir	Control Measures
Plague	Yersinia pestis	Bite by flea	Wild rodents	Control rodent populations, immunization
Rocky Mountain spotted fever	Rickettsia rickettsii	Bite by infected tick	Ticks, rabbits, mice	Avoid tick exposure; treat infected individuals with antibiotics
Psittacosis	Chlamydia psittaci	Contact with birds or bird excrement	Wild and domestic birds	Avoid contact with birds; treat infected individuals with antibiotic
Chlamydia	Chlamydia trachomatis	Urethral, vaginal, and anal secretions	Humans	Testing for organism during routine pelvic examinations; chemotherapy of carriers and potential contacts; case tracing and treatment
Gonorrhoea	Neisseria gonorrhoeae	Urethral and vaginal secretions	Humans	Chemotherapy of carriers and potential contacts; case tracing and treatment
Syphilis	Treponema pallidum	Infected exudates or blood	Humans	Identification by serologicall tests; antibiotic treatment of seropositive individuals
Meningicoccal meningitis	Neisseria meningitidis	Humans cases and carriers	Humans	Exposure treated with sulfadiazine for susceptible strains
Pneumococcal pneumonia	Streptococcus pneumoniae	Human carriers	Humans	Antibiotic treatment; isolation of cases for period of communicability
Tuberculosis	Mycobacterium tuberculosis	Sputum human cases; contaminated milk	Humans, cattle	Treatment with isoniazid; pasteurization of milk
Whooping cough	Bordetella pertussis	Human cases	Humans	Immunization; case isolation
Diphtheria	Corynebacterium diphtheriae	Human cases and carriers; infected food and fomites	humans	Immunization; quarantine of infected individuals
Legionnaire’s disease	Legionella pneumophila	Contaminated water	High moisture environments	Decontamination of air conditioning cooling towers, etc.
Paratyphoid	Salmonella paratyphi	Fecal contamination of food and water	Humans	Decontamination of public water sources; oversight of food handles; immunization
Typhoid fever	Salmonella typhi	Fecal contamination of food of food and water	Humans	Decontamination of public water sources; oversight of food handlers; pasteurization of milk; immunization
Tularemia	Salmonella typhi	Fecal contamination of food and water	Humans	Decontamination of public water sources; oversight of food handlers; pasteurization of milk; immunization
Anthrax	Bacillus anthracis	Milk or meat from infected animals	Cattle, swine, goats, sheep, horses	Destruction of infected animals
Bacillary dysentry	Shigella dysenteriae	Fecal contamination of food and water	Humans	Detection and control of carriers; oversight of food handlers; decontamination of water supplies
Botulism	Clostridium botulinum	Soil contaminated food	Soil	Proper preservation of milk; control of infection in animals
Brucellosis	Brucella melitensis	Milk or meat from infected animals	Cattle, swine, goats, sheep, horses	Pasteurization of milk; control of infection in animals
Cholera	Vibrio cholerae	Fecal contamination of food and water	Humans	Decontamination of public water sources; immunization
E. coli 0157 : H7 food infection	Escherichia coli O157 : H7	Fecal contamination of food and water	Humans, cattle	Decontamination of public water sources; oversight of food handlers; pasteurization of beverages
Epidemic typhus	Rickettsia prowazekii	Bite by infected louse	Humans, lice	Control louse Population
Lyme disease	Borrelia burgdorferi	Bite from infected tick	Rodents, deer, ticks	Avoid tick exposures; treat infected individuals with antibiotics