ABSTRACT
To combat the ever-increasing threat of wheat yellow rust worldwide, understanding of the pathogen (Puccinia striiformis) population biology is indispensable. Molecular markers, particularly microsatellites, have been reported to be important tools for deciphering pathogen population structure, invasion sources, and migration history. The utility of these DNA-based markers and sequencing has been increased by the direct DNA extraction from infected leaves with subsequent multiplex-based SSR genotyping. In this chapter we describe the protocol for direct DNA extraction and its genotyping with microsatellite markers in multiplex reactions. We describe the procedure for allele scoring, and various troubles faced during microsatellite scoring and potential solutions for them.
Subject(s)
Basidiomycota/genetics , DNA, Fungal/genetics , Genotyping Techniques/methods , Microsatellite Repeats , Plant Diseases/microbiology , Triticum/microbiology , Base Sequence , Chemical Fractionation/methods , DNA, Fungal/isolation & purification , Genotype , Polymerase Chain Reaction/methods , Spores, Fungal/geneticsABSTRACT
Avian Adeno viruses and Chicken Anemia Viruses cause serious economic losses to the poultry industry of Pakistan each year. Timely and efficient diagnosis of the viruses is needed in order to practice prevention and control strategies. In the first part of this study, we investigated broilers, breeder and Layer stocks for morbidity and mortality rates due to AAV and CAV infections and any co-infections by examining signs and symptoms typical of their infestation or post mortem examination. In the second part of the study, we developed a duplex PCR assay for the detection of AAV and CAV which is capable to simultaneously detect both the viral types prevalent in Pakistan with high sensitivity and 100% specificity.
Subject(s)
Adenoviridae Infections/diagnosis , Aviadenovirus/genetics , Chicken anemia virus/genetics , Chickens/virology , Circoviridae Infections/diagnosis , DNA, Viral/analysis , Multiplex Polymerase Chain Reaction/methods , Poultry Diseases/diagnosis , Adenoviridae Infections/epidemiology , Adenoviridae Infections/veterinary , Adenoviridae Infections/virology , Animals , Bone Marrow/chemistry , Circoviridae Infections/epidemiology , Circoviridae Infections/veterinary , Circoviridae Infections/virology , Coinfection , Pakistan , Poultry Diseases/epidemiology , Poultry Diseases/virology , Prevalence , Sensitivity and Specificity , Thymus Gland/chemistryABSTRACT
Hepatitis C is an infectious disease, caused by blood borne pathogen; the Hepatitis C Virus. In this study we analyzed blood samples collected from various risk groups for the prevalence of anti-HCV and active HCV infection with the help of Immunochromtographic tests and nested PCR. The prevalence of active HCV infection among the high risk groups was 15.57% (26/167). The prevalence of HCV in individual risk groups was 15%, 28%, 8%, 14.28% and 14.28% in the case of thalassemics, dialysis, major surgery group, dental surgery group and injection drug users respectively. Our analysis reveals the fact that health care facilities in the Khyber Pakhtunkhwa province of Pakistan are contributing a great deal towards the spread of HCV infection.
Subject(s)
Cross Infection/epidemiology , Hepacivirus/isolation & purification , Hepatitis C/epidemiology , Adolescent , Adult , Aged , Aged, 80 and over , Child , Cross Infection/virology , Female , Hepacivirus/genetics , Hepacivirus/immunology , Hepatitis C/virology , Hepatitis C Antibodies/blood , Humans , Immunoassay/methods , Male , Middle Aged , Pakistan/epidemiology , Polymerase Chain Reaction/methods , Prevalence , RNA, Viral/blood , Young AdultABSTRACT
BACKGROUND: Hepatitis C virus (HCV) genotype 3a is known to show comparatively better response to combination therapy than genotype 1 and 4. Mutations within NS5A gene of HCV have earlier been implicated with response to interferon (IFN) therapies in chronic HCV patients among various populations. As response to therapy are available in different populations because of the ethnic and viral factors and there was no study available on the phenomenon of resistivity to IFN. RESULTS: Chronic HCV 3a infected Pakistani patients were kept on IFN-α and ribavirin therapy for six months. NS5A gene of HCV was amplified and sequenced in the case of all the patients prior to therapy and the sequences were analysed for mutations. Out of the total 27 patients, 20 (74.07%) were observed with sustained virological response (SVR), 4 (14.81%) patients were non responder (NR) while 3 (11.11%) patients exhibited in end of treatment response (ETR). Three (3/20) (15%) SVR patients and two (2/3) ETR patients had mutations (ranging from I-V amino acids) within the NS5A ISDR regions. While the rest of the SVR patients (85%) and the NR had no mutations at ISDR region when compared with HCV K3a ISDR. CONCLUSIONS: Mutations within the NS5A gene of HCV 3a genotype may not influence the outcome of combination therapy in Pakistani populations.
Subject(s)
Antiviral Agents/administration & dosage , Hepacivirus/drug effects , Hepatitis C, Chronic/drug therapy , Viral Nonstructural Proteins/genetics , Adult , Amino Acid Substitution/genetics , Drug Therapy, Combination/methods , Female , Genotype , Hepacivirus/genetics , Hepacivirus/isolation & purification , Humans , Interferon-alpha/administration & dosage , Male , Middle Aged , Molecular Sequence Data , Mutation, Missense , Pakistan , RNA, Viral/genetics , Ribavirin/administration & dosage , Sequence Analysis, DNAABSTRACT
Hepatitis C is a fatal liver disease caused by the hepatitis C virus. In this study, blood donors, from various districts of the KPK province and the federally administered tribal area (FATA) of Pakistan were tested for anti-HCV antibodies and HCV RNA by ICT (Immuno-chromatographic test), ELISA and RT-PCR. Out of the 7148 blood donors, 224 (3.13%) were positive for anti-HCV antibodies by ICT, 135 (1.89%) by ELISA while 118 (1.65%) blood donors had active HCV infection as detected by RT-PCR. We suggest that ELISA should be used for anti-HCV screening in public sector hospitals and health care units.
