9 research outputs found
Sensitive detection of Mycobacterium bovis in spiked milk using polymerase chain reaction assay
Get PDFBovine tuberculosis is a chronic zoonotic disease that affects both animal and human health and imposes serious public health concerns in the world. Intake of non-pasteurized milk is considered the most probable vehicle for the transmission of pathogenic bacteria. In this study, the detection of Mycobacterium bovis BCG in spiked milk using a polymerase chain reaction was performed. The performance of two DNA extraction methods, CTAB/phenol:chloroform:isoamyl alcohol and EXTRAGENMB were also evaluated. In addition, Mycobacterial concentration was tried to determine using the Standard/ Viable Plate Count Method and Spectrophotometric (Turbidimetric) Method. PCR successfully detected M. bovis BCG in spiked milk, detecting approximately up to two bacilli per reaction. The two DNA extraction methods were effective in the isolation of amplifiable DNA, having the advantage of EXTRAGENMB in terms of (1) shorter duration of DNA extraction, (2) less sample manipulation, and (3) ease of execution of the procedure. Quantitative determination of the Mycobacterial population however failed to quantify the bacterial concentration per dilution, suggesting that CFU concentration should be considered an approximation. It is expected that this method can be used for the detection of M. bovis in raw milk samples
A New Loop-Mediated Isothermal Amplification Method for Rapid, Simple, and Sensitive Detection of Leptospira spp. in Urine
Get PDFWe developed a new loop-mediated isothermal amplification (LAMP) method to detect rrs, a 16S rRNA gene, of pathogenic Leptospira spp. in urine. The method enables detection of two leptospiral cells per reaction following boiling of urine specimens. The sensitivity of this method is higher than that of culture or of flaB-nested PCR
TRYPANOSOMA EVANSI AND NEOSPORA CANINUM AMONG WATER BUFFALOES (BUBALUS BUBALIS) IN THE PHILIPPINES
Get PDFThe study determined the positivity rate of Trypanosoma evansi and Neospora caninum antibodies in water buffaloes in the province of Nueva Ecija, Philippines using Polymerase Chain Reaction (PCR) for T. evansi and competitive Enzyme-linked Immunosorbent Assay (cELISA) for N. caninum antibodies . A total of 100 whole blood and 100 serum samples were collected to test for T. evansi and N. caninum , respectively. Rotat 1.2 VSG gene was target using PCR for T. evansi detection. Neospora caninum antibody detection was done from the serum samples using cELISA test kit.Results revealed that the positivity rate of T. evansi in Nueva Ecija was 11% (11/100). The positive animals identified were from the municipalities of Muñoz (4/16; 25%), Sta. Rosa (3/13; 23.08%) and Talugtug (4/16; 25%). The seropositive rate of Nueva Ecija for N. caninum. was 46% (46/100), seropositive animals were identified in Cabanatuan City, 57.14% (4/7); Science City of Muñoz, 43.14% (22/51); Sta. Rosa, 40% (4/10); Sto. Sunday, 50% (6/12); and Talugtug 50% (10/20). The seropositivity rate of N. caninum and the presence of T. evansi in Nueva Ecija may contribute to the cases of abortions in the province and further studies should be employed to confirm the association of these organisms to abortion cases on water buffaloes
Evaluation of LAMP for detection and/or screening of Leptospira spp. infection among domestic animals in the Philippines
No full textObjective: This study assessed the applicability of loop-mediated isothermal amplification (LAMP) for the detection of leptospirosis among domesticated animals and sewage rats. Specifically, it evaluated the ability of LAMP to amplify Leptospira spp. targeting the 16s rRNA gene in boiled urine samples.
Materials and methods: A total of 140 samples from different domestic animals were tested for the presence of the antigen. A nested-polymerase chain reaction (nPCR) protocol was used to compare and determine the sensitivity of LAMP in detecting Leptospira spp. The LAMP was also evaluated by comparing its amplification result using agarose gel electrophoresis and color change using dye.
Results: Positivity rate of Leptospira spp. antigen was 29.0% (40/140) for LAMP and 9.3% (13/140) for nPCR. Also, LAMP results for gel electrophoresis and dye color change varied in some samples that may be due to the interpretation of the result in dye color change.
Conclusion: Overall, LAMP is a rapid, sensitive, and cost-effective diagnostic method compared with nPCR. Also, LAMP has a potential application as pen-side screening, surveillance, and clinical diagnostic kits of infectious diseases without requiring advance equipment and skilled personnel. [J Adv Vet Anim Res 2018; 5(4.000): 459-465
A New Loop-Mediated Isothermal Amplification Method for Rapid, Simple, and Sensitive Detection of Leptospira spp. in Urine
No full textGenotyping and molecular detection of polymorphism in FUT1 gene of swine
Get PDFAlpha (1, 2)-fucosyltransferase (FUT1), as a candidate gene in controlling the expression of Escherichia coli F18 receptor has been identified to determine whether an animal (i.e., Sus scrofa, swine) is resistant or susceptible to enterotoxigenic E. coli (ETEC) infections. This study was conducted to determine the genotypes of 150 blood samples of three swine breeds. From these, 20 individuals were randomly selected for sequencing. Polymerase chain reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) results revealed that among the genotypes, seven of the Duroc x Pietrain samples carried the AG genotype which was previously reported to be susceptible to ETEC infection. Two AA genotypes were presumptively resistant and 50 that were presumptively susceptible, as these samples carried either AG or GG genotypes from the Landrace variety. Of the Large White samples, two samples carried the AA genotype and 89 had either AG or GG genotypes. Allelic frequency of allele A was 0.273, while, and allele G was 0.726. The DNA sequences registered 100% homologies to the S. scrofa FUT1 gene. Twelve of the sequenced samples exhibited a shift from G to A in the 117th nucleotide and one sample had a C to T shift in the 39th nucleotide. A change in the protein was observed from alanine to threonine at the 117th nucleotide position indicating a functional mutation. The FUT1 mutation was found in all the Duroc X Pietrain samples, as well as in samples with the AA genotype (Large White samples). Heterozygous forms of Landrace and Large White also exhibited this mutation. The constructed phylogenetic tree revealed 2 groupings based on the mutations at the 117th nucleotide
SCREENING OF THE ACID MEAT CONDITION IN THE RENDEMENT NAPOLE GENE USING POLYMERASE CHAIN REACTION - RESTRICTION FRAGMENT LENGTH POLYMORPHISM
Get PDFA mutation in the rendement napole (RN) gene causes the acid meat condition which results to poor meat quality due to its reduced water holding capacity, low pH, pale color, reduced processing and cooking yield due to increased drip, and strong metallic taste. This study was conducted to detect the mutation in the RN gene in 535 commercial breeder pigs from the Philippines. Blood collection was done then subjected to DNA extraction and genotyping using polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) using the enzyme BsrBI, then validated by DNA sequencing. Results revealed that 97.01% of the breeder pigs did not have the mutation in their RN gene, while 2.69% had at least one copy of the defective allele in their gene. The acid meat condition has only been previously detected in the Hampshire breed whereas this study found the mutations predominantly in Pietrain and Landrace breed they were classified as normal (rn/rn), heterozygous mutants (RN/rn), and homozygous mutants (RN/RN) which allowed breeding systems to be developed ensuring that all offspring are free of the defect. This genetic screening will help in detecting the presence of the defect in a given swine population and reduce the unwanted effects on meat quality thus increasing its market value
