TITLE: Brucella species detection from blood and milk samples
AUTHORS: Rania Baleela, Zienab Adam Ahmed Abdallh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">Brucellosis is a widespread zoonotic disease which is characterized by </div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">reduced fertility and abortion in several species of animals and in human.</div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Camels are highly susceptible to </span><span style = "font-style:italic;">Brucella abortu</span><span>, and </span><span style = "font-style:italic;">Brucella melitensis</span><span> but they are not the primary hosts.  </span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Consumption of </span><span style = "font-style:italic;">Brucella</span><span>infected milk and meat from camels is a serious public health issue though. Therefore rapid detection and diagnosis of </span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">Brucellosis is crucial for any control program.</div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>In this study, a sensitive and specific PCR assay was adapted for the detection and identification of </span><span style = "font-style:italic;">Brucellaspp</span><span>. in100 camels. Almost half of the collected samples were positive for </span><span style = "font-style:italic;">Brucellaspp</span><span>. (i. e. 41%).  44% of the samples were positive for Brucella abortus, 43% were positive for</span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span style = "font-style:italic;">B</span><span style = "font-style:italic;font-style:italic;">.</span><span style = "font-style:italic;"> melitensis</span><span>, and 22% were positive for a co-infection of </span><span style = "font-style:italic;">B. abortus</span><span> and</span><span style = "font-style:italic;">B.melitensis</span><span>.The majority of infected camels were male adults.Most of </span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">the positive samples were collected at the slaughterhouse in Nyala. This </div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">finding is alarming as Sudanese consumers are known for eating raw </div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">camels' liver. Therefore the main recommendation ofthis study is toadopt this sensitive and species-specific PCR protocol for routine </div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">detection of  brucellosis in animals to be slaughtered in the future . </div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">In the mean time this protocol can be used as a confirmatory test for the </div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">detection ofbrucellosis in live camels as  well as for other animals to be slaughtered because it is the protocol with the lowest risk for laboratory </div><style>
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[STEPS]
?. [DNA extraction:  The phenol / chloroform method was used to extract genomic DNA.]
Wash 1ml of blood with Red Blood Cells lysis buffer (NaHCO3 0.0841g, NH4Cl6.15135g 1000ml distilled water) twice centrifuge at 60000 rpm for 5min
?. [DNA extraction:  The phenol / chloroform method was used to extract genomic DNA.]
Same protocol is used to extract DNA from filter paper samples: add three 1cm pieces to 500µl lysis buffer (15.76gTris–HCl (pH 8), 10% SDS, and 1.8612gEDTA in 1000ml distilled water) leave overnight at 60 °C.
?. [DNA extraction:  The phenol / chloroform method was used to extract genomic DNA.]
Add 500µl of Phenol/Chloroform/Isomyalcohol (25:24:1)  to each sample then centrifuge for 5mn at 10000rpm ,
?. [DNA extraction:  The phenol / chloroform method was used to extract genomic DNA.]
Transfer supernatant to another  tube and add 500µl of Chloroform/Isomyalcohol (24:1)
?. [DNA extraction:  The phenol / chloroform method was used to extract genomic DNA.]
Centrifuge at 100000rpm for 5min.
?. [DNA extraction:  The phenol / chloroform method was used to extract genomic DNA.]
Add equal volume of 100 % ice cold ethanol and leave at -20°C overnight.
?. [DNA extraction:  The phenol / chloroform method was used to extract genomic DNA.]
Centrifuge precipitated DNA at 14000 rpm for 10 min and wash with 70% ethanol two times.
?. [DNA extraction:  The phenol / chloroform method was used to extract genomic DNA.]
Discard the supernatant and leave the tubes up- side-down to allow precipitated DNA to dry for 40min inside an incubator.
?. [DNA extraction:  The phenol / chloroform method was used to extract genomic DNA.]
Dissolve the extracted DNA in 100µl double distilled water;
?. [DNA extraction:  The phenol / chloroform method was used to extract genomic DNA.]
Quantify the concentration and store at -20°C.
?. [DNA extraction:  The phenol / chloroform method was used to extract genomic DNA.]
Same protocol is used to extract DNA from milk samples except for the use of RBCS lysis buffer.
?. [Detection of Brucella spp:  For a total volume of 25 μl:]
5 μl iNtRONMaxime™ PCR premix
?. [Detection of Brucella spp:  For a total volume of 25 μl:]
2 µl each primer (step 15),
?. [Detection of Brucella spp:  For a total volume of 25 μl:]
2 μl template DNA
?. [Conditions]
95°C for 5 min;
?. [Conditions]
35 cycle:  95°C for min, 65 °C for 1min, 72°C for ,1 min extension at 72°C for 7 min,
?. Final hold at 4°C.
?. Brucella spp. Detection primers (adopted from Khamesipouretal., 2014)    B. abortus   Forward primer: GACGAACGGAATTTTTCCAATCCC   Reveres primer:TGCCGATCACTTAAGGGCCTTCAT   Expected molecular size   494bp    B. melitensis   Forward primer: AAATCGCGTCCTTGCTGGTCTGA   Reveres primer: TGCCGATCACTTAAGGGCCTTCAT   Expected molecular size    743bp
?. [Conditions]
Preheat lid to110°C,
?. Visualization:  2% (w/v) agarose gel containing 0.5× TBE buffer (743 mMTris–HCl (pH 8), 87 mM boric acid, and 5.3mM Na2EDTA), stained with ethidium bromide.
?. [DNA extraction:  The phenol / chloroform method was used to extract genomic DNA.]
Add 1ml lysis buffer and leave at 60°C  overnight.