ARBITRARY PRIMED PCR
The Arbitrarily primed polymerase chain reaction (AP-PCR) is a type of PCR which is also known as Random Amplified Polymorphic DNA (RAPD). It is based on DNA fingerprinting technique in which primers are used whose nucleotide sequence is arbitrarily chosen.
After the invention of PCR it was realized that short primer would bind on several locations in a genome and thus could multiple fragments. In 1990 J. Welsh and McClelland uses long primers but lowers primer stringency for priming at many sites while William et.al also in 1990 develop RAPD technique used very short 10 base primer to generate random fragments from template DNA.
IMPORTANCE OF AP-PCR
It is observed that for a determined set of experimental conditions the pattern of DNA bands that was amplified are unique and reproducible. While by the comparison with other DNA fingerprinting approaches, the AP-PCR method allows direct cloning of the in-vitro amplified DNA sequences.
Two main important aspects of this method are as follows:
- The amplified bands usually but not always originate from single copy sequences.
- The amplification is quantitative i.e. the intensities of the amplified bands are almost proportional to the concentration of the corresponding target sequences in the DNA preparation.
From a single arbitrary primer, large number of bands are amplified which form a complex fingerprint that can be utilized to detect relative differences in the arbitrarily amplified DNA sequences from two different but closely related genomes. These differences are comparable polymorphisms or somatic genetic alterations. The AP-PCR shows multiple applications, including genetic mapping of polymorphisms, taxonomy and detection of somatic mutations in cancer cells.
PATHOGEN DETECTION USING AP-PCR
Brucellosis is a worldwide zoonotic disease caused by member of the genus Brucella which affects a large range of domestic livestock, wildlife, marine mammals, and humans. The bacteria are transmitted from animals to humans by ingestion of infected food products or direct contact with an infected animal or by inhalation of aerosols. Brucellosis have been known by various names like Mediterranean fever, Malta fever, gastric remittent fever, and undulant fever. Humans are accidental hosts even though brucellosis considered major public health concern worldwide. The bacteria that cause infection is localized in the reproductive organs of host animals causing abortions and sterility. They are sheded in large numbers from the infected animal’s urine, milk, placental and other fluids.
Molecular based methods have provided powerful tools to identify infectious organisms with high sensitivity and specificity. From this article the molecular characterization of the isolated 1.3 kb DNA fragment and use of the sequence information from PCR based assays for the detection of Brucella DNA from spiked blood and milk samples and also from the organ tissues of infected animals.
The Brucella cultures were grown on potato agar, under conditions of 10% CO2 and 37 °C for 48 h. Cultures were suspended in sterile 0.01 M Tris, pH 7.3 and killed by heating at 80 °C for 30 min. The non-Brucella bacterial species were cultured according to ATCC media recommendations.
Genomic DNA isolation, PCR, cloning and sequencing of Brucella specific fragment:
- Before DNA extraction, the bacterial cultures were washed with PBS and pelleted by centrifugation. Then the total DNA were isolated from heat-killed Brucella cells and also the DNA from other bacterial species were also isolated.
- Sequence of the random primers used in the study were, OPB-01 (50 -GTTTCGCT CC-30), OPB-03 (50 -CATCCCCCTG-30), OPB-05 (50 -TGCGC CCTTC-30), OPB-19 (50 -ACCCCCGAAG-30) and OPA-20 (50 -GTTGCGATCC-30).
- A standard PCR amplification reaction mixture of 25 µl were contained in 5 ng genomic DNA, 1.5 mM MgCl2, 1 lM primer, 1.5 units DNA polymerase, and 200 lM deoxynucleoside triphosphate (dNTP), in 10 mM Tris/HCl, pH 8.8, 10 mM KCl, and 0.002% Tween-10.
- A negative control that contained all the components of the PCR reaction except the template DNA was included to monitor any possible contamination.
- PCR was performed in an ABI/Perkin Elmer 9700 thermo cycler. The program of 30 cycles consisted of 1 min at 94 °C, 2 min at 35 °C, and 3 min at 72 °C, and a final extension step at 72 °C for 7 min. Samples were held at 4 °C until electrophoresis.
- Following PCR amplification, the reaction mixture was analyzed on a 1.5% (w/v) agarose gel containing 0.5 lg/ml of ethidium bromide in TBE buffer (89 mM Tris/HCl, 89 mM boric acid, 2 mM EDTA, pH 8.0). A 100 bp DNA ladder was used as size standard.
- The band was excised and purified using gel extraction kit (Qiagen). Then the purified PCR fragment was inserted into a pCR2.1-TOPO vector according to the protocol described in the manual.
- The ligation mixture was used to transform E. coli TOP10F competent cells. Number of colonies were screened for the presence of 1.3 kb insert. One of the positive clones was sequenced from both strands. DNA sequence data were analyzed by comparison with the published sequences in the NCBI GenBank database using BLAST.
- Several internal primers were designed and screened to identify primer pairs that would amplify a smaller and more specific DNA fragment from Brucella sp. Sequence of selected internal primers are, KW1 (50 -CGGCTTGTTTCGCTCCA TCGG-30),KW2 (50 -GATTTCATTCAGCACGATACG-30), KW3 (50 -GCTTCGTGAACGGTGCGCTGG-30) and KW4 (50 -CGCACGGATTTCATTCTCTAC-30).
Preparation of spiked samples
The recovery of pathogen-DNA and sensitivity of the detection method was tested using milk and blood samples containing the pathogen DNA. Whole human blood was obtained from Kuwait Blood Bank used for spiking with Brucella DNA with citrate phosphate dextrose anticoagulant.
Total DNA isolation from milk was performed according to the methods. In both type of samples, Brucella DNA was added at the concentration range of 1 ng to 1 µg per ml. Total DNA was isolated from samples and PCR were performed for the detection of Brucella DNA.
Evaluation of the PCR based detection method for the presence of Brucella in sheep tissues:
Organs from healthy and infected sheep were obtained from a local abattoir. The DNA was isolated from liver, kidney and lymph tissues according the protocol. PCR was performed using primers KW3 and KW4.
Brucellosis is one of the major zoonotic diseases in the developing countries. Early detection of the disease is important for the effective control. Vaccination reduce losses due to Brucellosis related abortions and mortality. As the antibody based method used for diagnoses of brucellosis, this method is of low specificity due to the presence of identical antigens (lipopolysaccharide) in various other gram negative bacterial pathogens. Therefore the development of PCR based detection methods would greatly improve the efficiency of detection. ). For the development of an efficient PCR based detection method, a number of random primers have been tested to select a primer that can produce consistent amplification of Brucella specific DNA fragment. The DIG-labeled 1.3 kb DNA fragment was able to detect the nano-gram scale of genomic DNA from B. abortus biotype 1, B. abortus (strain S-19), B. melitensis biotype 1 and B. melitensis RebI. There were no cross reactivity signals observed from DNA samples of bacterial species in sheeps.
The study also shows that PCR based detection of Brucella from the samples of milk and blood spiked with known amount of the pathogen DNA. Sequencing of the Brucella specific DNA fragment revealed some interesting information. Based on the deduced amino acid sequence analysis, the isolated fragment belongs to D-alanine–D-alanine ligase gene. The sequence had no homology to published sequences reported from non-Brucella species at the nucleotide level.
This is one of the reasons for the high specificity of the PCR. Some degree of homology can be noticed only at the deduced amino acid level to D-alanine–Dalanine ligases of other bacterial species. As this PCR based method can be utilized effectively in the detection of Brucellosis. Degree of sensitivity of PCR based method is a key issue for its effective use in the diagnosis of Brucellosis. Internal primers were designed for use in the routine detection of Brucella infection. The results clearly show that KW3/KW4 produces consistent results from which Brucella can be detected. Furthermore, experiments on milk and blood samples spiked with Brucella DNA, clearly show a high sensitivity level of detection method.
In conclusion the application of the developed method at the field level, for the screening of the disease in animal tissues, demonstrate its great utility for the detection of the disease. PCR based method of detection is relatively rapid, specific and simple. In contrast with other DNA fingerprinting approaches, the AP-PCR method permits direct cloning of the in vitro amplified DNA sequences.
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