Objective:To assess the role of polymerase chain reaction(PCR)in serum sauples,in the diagnosis of osteoarticular tuberculosis(OTB)in a setting where only clinical and imaging diagnoses determine the treatment.Methods...Objective:To assess the role of polymerase chain reaction(PCR)in serum sauples,in the diagnosis of osteoarticular tuberculosis(OTB)in a setting where only clinical and imaging diagnoses determine the treatment.Methods:A total of 44 consecutive serum specimens were collected from clinically suspected OTB patients,based on clinical and radiological[X-ray or magnetic resonance imagng/computecl tomography]features.They were scrcened by in-house nested PCR.In addition,a few specimens were examined by Gram stain,acid-fast bacilli stain,histand routine bacterial culture.A total of 39 specimens were collected from patients suffering from other bone diseases of nontuberculous origin and included as negative controls.Results:of the 44 clinically suspected OTB patients,in-house nested PCR was positive in 40(91%)cases;PCR was negative in 38(97%)negative controls.Sensitivity and specificity of our in—house nested PCR was 90.3%and 97.4%,respectively.The PCR report was available within 48 h.It was possible to standardize serum PCR technique and in positive cases,a good n was observed in terms of an adequate treatment response.Conclusions:Nested PCR in serum samples is a rapid,highly sensitive and specific modality for OTB detection,PCR should be performed in addition to clinical evaluation,imaging studies,acidfast bacilli staining,culture and histopathology diagnosis,if possible.展开更多
To apply an individualized oncological approach to gastric cancer patients,the accurate diagnosis of disease entities is required.Peritoneal metastasis is the most frequent mode of metastasis in gastric cancer,and the...To apply an individualized oncological approach to gastric cancer patients,the accurate diagnosis of disease entities is required.Peritoneal metastasis is the most frequent mode of metastasis in gastric cancer,and the tumor-node-metastasis classification includes cytological detection of intraperitoneal cancer cells as part of the staging process,denoting metastatic disease.The accuracy of cytological diagnosis leaves room for improvement;therefore,highly sensitive molecular diagnostics,such as an enzyme immunoassay,reverse transcription polymerase chain reaction,and virusguided imaging,have been developed to detect minute cancer cells in the peritoneal cavity.Molecular targeting therapy has also been spun off from basic research in the past decade.Although conventional cytologyis still the mainstay,novel approaches could serve as practical complementary diagnostics to cytology in near future.展开更多
Hepatitis C virus(HCV) is an emerging infection worldwide and the numbers of persons infected are increasing every year. Poor blood transfusion methods along with unsafe injection practices are potential sources for t...Hepatitis C virus(HCV) is an emerging infection worldwide and the numbers of persons infected are increasing every year. Poor blood transfusion methods along with unsafe injection practices are potential sources for the rapid spread of infection. Early detection of HCV is the need of the hour especially in high riskgroup population as these individuals are severely immunocompromised. Enzyme Immunoassays are the most common detection techniques but they provide no evidence of active viremia or identification of infected individuals in the antibody-negative phase and their efficacy is limited in individuals within high risk group population. Molecular virological techniques have an important role in detecting active infection with utmost specificity and sensitivity. Technologies for assessment of HCV antibody and RNA levels have improved remarkably, as well as our understanding of how to best use these tests in patient management. This review aims to give an overview of the different serological and molecular methods employed in detecting HCV infection used nowadays. Additionally, the review gives an insight in the new molecular techniques that are being developed to improve the detection techniques particularly in High Risk Group population who are severely immunocompromised.展开更多
Objective:To establish a polymerase chain reaction(PCR) technique based on cytochrome b {cytb) gene of mitochondria DNA(mtDNA) for blood meal identification.Methods:The PCR technique was established based on published...Objective:To establish a polymerase chain reaction(PCR) technique based on cytochrome b {cytb) gene of mitochondria DNA(mtDNA) for blood meal identification.Methods:The PCR technique was established based on published information and validated using blood sample of laboratory animals of which their whole gene sequences are available in CenBank.PCR was next performed to compile gene sequences of different species of wild rodents.The primers used were complementary to the conserved region of the cytb gene of vertebrate's mtDNA.A total of 100 blood samples,both from laboratory animals and wild rodents were collected und analyzed.The obtained unknown sequences were compared with those in the GenBank database using BLAST program to identify the vertebrate animal species.Results:Gene sequences of 11 species of wild animals caught in 9 localities of Peninsular Malaysia were compiled using the established PCR. The animals involved were Rattus(rattus) tanezumi,Rattus tiomanicus,Leopoldamys sabanus, Tupaia glis,Tupaia minor,Niviventor cremoriventor,Rhinosciurus laticaudatus,Calloseiurus caniseps,Sundamys muelleri,Rattus rajah,and Maxomys whitelwadi.The BLAST results confirmed the host with exact or nearly exact matches(>89%identity).Ten new gene sequences have been deposited in CenBank database since September 2010.Conclusions:This study indicates that the PCR direct sequencing system using universal primer sets for vertebrate cytb gene is a promising technique for blood meal identification.展开更多
BACKGROUND Histopathologically stained archived tissue slides are stored in hospital archives for years to decades.They are the largest available source of biological materials and are a potentially useful resource th...BACKGROUND Histopathologically stained archived tissue slides are stored in hospital archives for years to decades.They are the largest available source of biological materials and are a potentially useful resource that can be used for retrospective epidemiological studies.DNA recovered from the slides can be used for several downstream molecular processes including polymerase chain reaction,single nucleotide polymorphism analysis,and whole genome sequencing.The DNA from these slides can be utilized to compare gene signatures of normal and diseased tissues.However,extraction of high-quality DNA from archived stained hematoxylin and eosin(H&E)slides remains challenging.AIM To standardize a new protocol for extracting DNA from archived H&E-stained tissue slides for further molecular assays.METHODS A total of 100 archived H&E-stained cancer slides were subjected to a total of five methods of DNA extraction.Methods were varied in the deparaffinization step,tissue rehydration,duration of lysis,and presence or absence of proteinase K.The extracted DNA was quantified using a NanoDrop spectrophometer and the quality was analyzed by agarose gel electrophoresis.Then each sample was subjected to polymerase chain reaction(PCR)to amplify the internal control gene GAPDH,thereby confirming the DNA intactness,which could be further utilized for other downstream applications.RESULTS Of the five different methods tested,the third method wherein xylene was used for tissue deparaffinization followed by 72 h of digestion and without proteinase K inactivation yielded the highest amount of DNA with good purity.The yield was significantly higher when compared to other methods.In addition,90%of the extracted DNA showed amplifiable GAPDH gene.CONCLUSION Here we present a step-by-step,cost-effective,and reproducible protocol for the extraction of PCR-friendly DNA from archived H&E-stained cancer tissue slides that can be used for further downstream molecular applications.展开更多
Infectious Bronchitis (IB) is highly contagious disease of commercial poultry causing substantial economic loses by producing poor quality meat in broilers and effecting production in breeder birds. The causative agen...Infectious Bronchitis (IB) is highly contagious disease of commercial poultry causing substantial economic loses by producing poor quality meat in broilers and effecting production in breeder birds. The causative agent has been reported as most hazardous pathogen among other infectious agent even after being immunized with multi-variant strain vaccine. Currently, different strain such as H-120, 4/91 and D274 have been used extensively for immunoprophylaxis against velogenic strain across Pakistan with minimal protection reported. In current study PCR analysis was used to investigate the molecular nature of IB isolates from Punjab and Sind province of Pakistan in 2016 epidemics. Total of 100 tracheal samples were considered for virus inoculation in 10 days old chicken embryonated eggs. The IBV infected amniotic fluid was neutralized with monoclonal antisera of H-120, 4/91 and D274 strains. The IBV screened samples were subjected for RNA extraction and subsequent to PCR using type specific primer of each strain. The amplified product of 840 bp was sequenced through Sanger sequencing. On the basis of PCR results, four similar amplified products from both regions were obtained showing similarities in agarose gel electrophoresis, but they differ from each other on the basis of nucleotides sequence. Phylogenetic analysis revealed that nucleotide sequences of isolates from Karachi were similar to the IBV H-120, Mass-41 and Connecticut 46 reference strains. Whereas, isolates from the Punjab province are analogous to the Mans-2, Mans-3, 9/41(UK) but did not show significant similarity with other reference strain. Therefore, it is recommended that use of M-41 and H-120 in vaccine production could be effective measure against velogenic infectious agent in Sindh particularly in Karachi, whereas, it would be better to incorporate either of the variant GQ281656.1, AY279533.1 in vaccine because of their highest level of resemblance with genetically sequenced isolates from Lahore and its surroundings.展开更多
The pattern of clinical forms of respiratory tuberculosis in children shows a preponderance of intrathoracic lymph node tuberculosis （89.4%） that is characterized by a complicated process in every third child under ...The pattern of clinical forms of respiratory tuberculosis in children shows a preponderance of intrathoracic lymph node tuberculosis （89.4%） that is characterized by a complicated process in every third child under present-day conditions. Positive result of PCR closely correlates with the severity and extent of the specific process in children. Real-time PCR （RT-PCR） was ascertain to exhibit the highest sensitivity in detecting Mycobacterium tuberculosis DNA in children with primary generalized tuberculosis （62.5%） and in those with a disseminated specific process （55.6%）, which was much higher than conventional bacteriological study of diagnostic materials. By taking into account the findings, the RT-PCR detection of M. tuberculosis was considered as a substantial criterion for evaluating the magnitude of specific changes and the degree of tuberculosis infection activity in children.展开更多
Coronavirus disease-19(COVID-19)has become a pandemic,being a global health concern since December 2019 when the first cases were reported.Severe acute respiratory syndrome coronavirus 2,the COVID-19 causal agent,is a...Coronavirus disease-19(COVID-19)has become a pandemic,being a global health concern since December 2019 when the first cases were reported.Severe acute respiratory syndrome coronavirus 2,the COVID-19 causal agent,is aβ-coronavirus that has on its surface the spike protein,which helps in its virulence and pathogenicity towards the host.Thus,effective and applicable diagnostic methods to this disease come as an important tool for the management of the patients.The use of the molecular technique PCR,which allows the detection of the viral RNA through nasopharyngeal swabs,is considered the gold standard test for the diagnosis of COVID-19.Moreover,serological methods,such as enzyme-linked immunosorbent assays and rapid tests,are able to detect severe acute respiratory syndrome coronavirus 2-specific immunoglobulin A,immunoglobulin M,and immunoglobulin G in positive patients,being important alternative techniques for the diagnostic establishment and epidemiological surveillance.On the other hand,reverse transcription loop-mediated isothermal amplification also proved to be a useful diagnostic method for the infection,mainly because it does not require a sophisticated laboratory apparatus and has similar specificity and sensitivity to PCR.Complementarily,imaging exams provide findings of typical pneumonia,such as the ground-glass opacity radiological pattern on chest computed tomography scanning,which along with laboratory tests assist in the diagnosis of COVID-19.展开更多
文摘Objective:To assess the role of polymerase chain reaction(PCR)in serum sauples,in the diagnosis of osteoarticular tuberculosis(OTB)in a setting where only clinical and imaging diagnoses determine the treatment.Methods:A total of 44 consecutive serum specimens were collected from clinically suspected OTB patients,based on clinical and radiological[X-ray or magnetic resonance imagng/computecl tomography]features.They were scrcened by in-house nested PCR.In addition,a few specimens were examined by Gram stain,acid-fast bacilli stain,histand routine bacterial culture.A total of 39 specimens were collected from patients suffering from other bone diseases of nontuberculous origin and included as negative controls.Results:of the 44 clinically suspected OTB patients,in-house nested PCR was positive in 40(91%)cases;PCR was negative in 38(97%)negative controls.Sensitivity and specificity of our in—house nested PCR was 90.3%and 97.4%,respectively.The PCR report was available within 48 h.It was possible to standardize serum PCR technique and in positive cases,a good n was observed in terms of an adequate treatment response.Conclusions:Nested PCR in serum samples is a rapid,highly sensitive and specific modality for OTB detection,PCR should be performed in addition to clinical evaluation,imaging studies,acidfast bacilli staining,culture and histopathology diagnosis,if possible.
基金Supported by Grants from Ministry of Education,Science,and Culture,Japan(to Kagawa S)and JSPS KAKENHI,No.23591932
文摘To apply an individualized oncological approach to gastric cancer patients,the accurate diagnosis of disease entities is required.Peritoneal metastasis is the most frequent mode of metastasis in gastric cancer,and the tumor-node-metastasis classification includes cytological detection of intraperitoneal cancer cells as part of the staging process,denoting metastatic disease.The accuracy of cytological diagnosis leaves room for improvement;therefore,highly sensitive molecular diagnostics,such as an enzyme immunoassay,reverse transcription polymerase chain reaction,and virusguided imaging,have been developed to detect minute cancer cells in the peritoneal cavity.Molecular targeting therapy has also been spun off from basic research in the past decade.Although conventional cytologyis still the mainstay,novel approaches could serve as practical complementary diagnostics to cytology in near future.
文摘Hepatitis C virus(HCV) is an emerging infection worldwide and the numbers of persons infected are increasing every year. Poor blood transfusion methods along with unsafe injection practices are potential sources for the rapid spread of infection. Early detection of HCV is the need of the hour especially in high riskgroup population as these individuals are severely immunocompromised. Enzyme Immunoassays are the most common detection techniques but they provide no evidence of active viremia or identification of infected individuals in the antibody-negative phase and their efficacy is limited in individuals within high risk group population. Molecular virological techniques have an important role in detecting active infection with utmost specificity and sensitivity. Technologies for assessment of HCV antibody and RNA levels have improved remarkably, as well as our understanding of how to best use these tests in patient management. This review aims to give an overview of the different serological and molecular methods employed in detecting HCV infection used nowadays. Additionally, the review gives an insight in the new molecular techniques that are being developed to improve the detection techniques particularly in High Risk Group population who are severely immunocompromised.
基金financially supported by a grant(JPP-IMR Code:09-030) from the Ministry of Health,Malaysia
文摘Objective:To establish a polymerase chain reaction(PCR) technique based on cytochrome b {cytb) gene of mitochondria DNA(mtDNA) for blood meal identification.Methods:The PCR technique was established based on published information and validated using blood sample of laboratory animals of which their whole gene sequences are available in CenBank.PCR was next performed to compile gene sequences of different species of wild rodents.The primers used were complementary to the conserved region of the cytb gene of vertebrate's mtDNA.A total of 100 blood samples,both from laboratory animals and wild rodents were collected und analyzed.The obtained unknown sequences were compared with those in the GenBank database using BLAST program to identify the vertebrate animal species.Results:Gene sequences of 11 species of wild animals caught in 9 localities of Peninsular Malaysia were compiled using the established PCR. The animals involved were Rattus(rattus) tanezumi,Rattus tiomanicus,Leopoldamys sabanus, Tupaia glis,Tupaia minor,Niviventor cremoriventor,Rhinosciurus laticaudatus,Calloseiurus caniseps,Sundamys muelleri,Rattus rajah,and Maxomys whitelwadi.The BLAST results confirmed the host with exact or nearly exact matches(>89%identity).Ten new gene sequences have been deposited in CenBank database since September 2010.Conclusions:This study indicates that the PCR direct sequencing system using universal primer sets for vertebrate cytb gene is a promising technique for blood meal identification.
基金the junior research fellowship from the Council of Scientific and Industrial Research, Government of India
文摘BACKGROUND Histopathologically stained archived tissue slides are stored in hospital archives for years to decades.They are the largest available source of biological materials and are a potentially useful resource that can be used for retrospective epidemiological studies.DNA recovered from the slides can be used for several downstream molecular processes including polymerase chain reaction,single nucleotide polymorphism analysis,and whole genome sequencing.The DNA from these slides can be utilized to compare gene signatures of normal and diseased tissues.However,extraction of high-quality DNA from archived stained hematoxylin and eosin(H&E)slides remains challenging.AIM To standardize a new protocol for extracting DNA from archived H&E-stained tissue slides for further molecular assays.METHODS A total of 100 archived H&E-stained cancer slides were subjected to a total of five methods of DNA extraction.Methods were varied in the deparaffinization step,tissue rehydration,duration of lysis,and presence or absence of proteinase K.The extracted DNA was quantified using a NanoDrop spectrophometer and the quality was analyzed by agarose gel electrophoresis.Then each sample was subjected to polymerase chain reaction(PCR)to amplify the internal control gene GAPDH,thereby confirming the DNA intactness,which could be further utilized for other downstream applications.RESULTS Of the five different methods tested,the third method wherein xylene was used for tissue deparaffinization followed by 72 h of digestion and without proteinase K inactivation yielded the highest amount of DNA with good purity.The yield was significantly higher when compared to other methods.In addition,90%of the extracted DNA showed amplifiable GAPDH gene.CONCLUSION Here we present a step-by-step,cost-effective,and reproducible protocol for the extraction of PCR-friendly DNA from archived H&E-stained cancer tissue slides that can be used for further downstream molecular applications.
文摘Infectious Bronchitis (IB) is highly contagious disease of commercial poultry causing substantial economic loses by producing poor quality meat in broilers and effecting production in breeder birds. The causative agent has been reported as most hazardous pathogen among other infectious agent even after being immunized with multi-variant strain vaccine. Currently, different strain such as H-120, 4/91 and D274 have been used extensively for immunoprophylaxis against velogenic strain across Pakistan with minimal protection reported. In current study PCR analysis was used to investigate the molecular nature of IB isolates from Punjab and Sind province of Pakistan in 2016 epidemics. Total of 100 tracheal samples were considered for virus inoculation in 10 days old chicken embryonated eggs. The IBV infected amniotic fluid was neutralized with monoclonal antisera of H-120, 4/91 and D274 strains. The IBV screened samples were subjected for RNA extraction and subsequent to PCR using type specific primer of each strain. The amplified product of 840 bp was sequenced through Sanger sequencing. On the basis of PCR results, four similar amplified products from both regions were obtained showing similarities in agarose gel electrophoresis, but they differ from each other on the basis of nucleotides sequence. Phylogenetic analysis revealed that nucleotide sequences of isolates from Karachi were similar to the IBV H-120, Mass-41 and Connecticut 46 reference strains. Whereas, isolates from the Punjab province are analogous to the Mans-2, Mans-3, 9/41(UK) but did not show significant similarity with other reference strain. Therefore, it is recommended that use of M-41 and H-120 in vaccine production could be effective measure against velogenic infectious agent in Sindh particularly in Karachi, whereas, it would be better to incorporate either of the variant GQ281656.1, AY279533.1 in vaccine because of their highest level of resemblance with genetically sequenced isolates from Lahore and its surroundings.
文摘The pattern of clinical forms of respiratory tuberculosis in children shows a preponderance of intrathoracic lymph node tuberculosis （89.4%） that is characterized by a complicated process in every third child under present-day conditions. Positive result of PCR closely correlates with the severity and extent of the specific process in children. Real-time PCR （RT-PCR） was ascertain to exhibit the highest sensitivity in detecting Mycobacterium tuberculosis DNA in children with primary generalized tuberculosis （62.5%） and in those with a disseminated specific process （55.6%）, which was much higher than conventional bacteriological study of diagnostic materials. By taking into account the findings, the RT-PCR detection of M. tuberculosis was considered as a substantial criterion for evaluating the magnitude of specific changes and the degree of tuberculosis infection activity in children.
文摘Coronavirus disease-19(COVID-19)has become a pandemic,being a global health concern since December 2019 when the first cases were reported.Severe acute respiratory syndrome coronavirus 2,the COVID-19 causal agent,is aβ-coronavirus that has on its surface the spike protein,which helps in its virulence and pathogenicity towards the host.Thus,effective and applicable diagnostic methods to this disease come as an important tool for the management of the patients.The use of the molecular technique PCR,which allows the detection of the viral RNA through nasopharyngeal swabs,is considered the gold standard test for the diagnosis of COVID-19.Moreover,serological methods,such as enzyme-linked immunosorbent assays and rapid tests,are able to detect severe acute respiratory syndrome coronavirus 2-specific immunoglobulin A,immunoglobulin M,and immunoglobulin G in positive patients,being important alternative techniques for the diagnostic establishment and epidemiological surveillance.On the other hand,reverse transcription loop-mediated isothermal amplification also proved to be a useful diagnostic method for the infection,mainly because it does not require a sophisticated laboratory apparatus and has similar specificity and sensitivity to PCR.Complementarily,imaging exams provide findings of typical pneumonia,such as the ground-glass opacity radiological pattern on chest computed tomography scanning,which along with laboratory tests assist in the diagnosis of COVID-19.
