scholarly journals Evaluation of Nested Polymerase Chain Reaction targeting hsp65 of Mycobacterium tuberculosis for the Detection of Organism in the Sputum Samples

2016 ◽  
Vol 20 (1) ◽  
pp. 28-33
Author(s):  
Yogaraje GC Varadaiah ◽  
Raghu K Chinnappa ◽  
Rakshitha M Nagaraj ◽  
Thejashwini NA ◽  
Robinson K Samuel ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Mycobacterium Tuberculosis ◽  
Negative Predictive Value ◽  
Nested Pcr ◽  
Predictive Value ◽  
Healthy Controls ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Gene Target ◽  
Polymerase Chain

ABSTRACT Introduction The poor sensitivity of conventional smear microscopy and the delay in obtaining Mycobacterium culture results prevent the early diagnosis of Myobacterium tuberculosis (MTB). By using nucleic acid amplification techniques like polymerase chain reaction (PCR), one may be able to diagnose the disease on the day of arrival of specimen in the laboratory. The present study aimed to evaluate the applicability of the nested-PCR (nPCR) technique as a rapid and direct molecular method for the diagnosis of M. tuberculosis in sputum specimens of patients whose sputum smear was acid-fast bacilli (AFB) negative using heat shock protein (hsp65) as the gene target. Materials and methods Early morning sputum samples were collected in sterile containers respectively from about 40 suspected patients of pulmonary tuberculosis, attending the outpatient units of JSS Medical College and PKTB Hospital, Mysore and from 20 age and sex-matched healthy controls. Sputum samples were decontaminated by modified Petroff's method and DNA was isolated using QIAGEN DNA extraction kit. The nPCR was carried out for the detection of MTB using the target gene hsp65. Results Nested-PCR showed specific amplification (165bp) of M. tuberculosis in 18 out of 20 sputum AFB positive samples and 9 out of 20 AFB negative samples. None of the healthy controls showed any amplification with nPCR. The nPCR when compared to that of Ziehl-Neelsen staining had a sensitivity of 90%, specificity of 77.5%, positive predictive value (PPV) of 66.6%, and negative predictive value (NPV) of 93.9%. The percentage of false positive was 33.3% and percentage of false negative was 6.1%. Conclusion The detection of M. tuberculosis with nPCR in smear negative patients provides the bacteriological data 4 to 8 weeks earlier. A molecular approach, based on the amplification of hsp65 gene by nPCR, showed that there is high probability of the disease being absent when the test is negative because of the high negative predictive value (NPV). How to cite this article Varadaiah YGC, Prashant A, Chinnappa RK, Nagaraj RM, Thejashwini, Samuel RK, Devegowda D, Vishwanath P. Evaluation of Nested Polymerase Chain Reaction targeting hsp65 of Mycobacterium tuberculosis for the Detection of Organism in the Sputum Samples. Indian J Med Biochem 2016;1(1):28-33.

Epidemiology of Epizootic Lymphangitis of cart horses in Northern Ethiopia Using Conventional diagnostic Methods and Nested Polymerase Chain Reaction

2019 ◽  
Author(s):  
Birhanu Hadush Abera ◽  
Molla Michaelay ◽  
Habtamu Taddele ◽  
Nigus Abebe ◽  
Abrha Tesfay ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Clinical Examination ◽  
Roc Curve ◽  
Nested Pcr ◽  
Control Measure ◽  
Diagnostic Methods ◽  
Northern Ethiopia ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Polymerase Chain

Abstract Background: Epizootic lymphangitis (EL), caused by Histoplasma capsulatum variety farciminosum (HCF) is a contagious chronic disease of equines characterized by development of nodular lesions in the lymph nodes, lymphatic vessels and skin. This disease is the most important diseases of equines in Ethiopia causing a significant economic loss, particularly cart pulling equines. Todate there is no sound diagnostic nor control measure implemented in the country. Furthermore, there is a shortage of data on the epidemiology of the disease in different regions of the country including northern Ethiopia. This study was conducted to investigate the epidemiology of EL in northern Ethiopia using the conventional methods and the nested polymerase chain reaction (PCR). Methods: A total of 191 cart-horses were enrolled and used as sources of pus and blood samples. The blood was used for the extraction of the DNA of HCF from buffy coat for nested PCR while the pus samples were cultured on Sabourauds Dextrose Agar for isolation. Statistical Package for Social Sciences (SPSS) version 21 was used for data analysis by applying logistic regression, receiver operating characteristic (ROC) curve and Cohen’s kappa coefficient test. In addition, the level of agreement between the clinical examination and the nested PCR was evaluated. Results: Infection with HCF was confirmed in 44% (84/191) of the horses using nested PCR. Subclinical infection was observed in 18.18% (22/121) of the apparently healthy horses. Considering nested PCR as a gold standard, the sensitivity and specificity of the clinical examination were 74% and 95%, respectively while the area under the ROC curve (AUR) was 0.83 (95% confidence interval, 0.77, 0.896). Moreover, a moderate (k=0.675) agreement was observed between the nested PCR and clinical examination.Conclusions: The findings of the present study showed the wide spread occurrence of EL in northern Ethiopia and the advantage of the nested PCR in detecting of the infection of HCF even before the clinical symptoms are apparent.

scholarly journals In SituReverse Transcriptase-Nested Polymerase Chain Reaction to Identify Intracellular Nucleic Acids without the Necessity of Dnase Pretreatment and Hybridisation

2001 ◽  
Vol 22 (3) ◽  
pp. 151-158 ◽  
Author(s):  
Mario Menschikowski ◽  
Margot Vogel ◽  
Rolf Eckey ◽  
Gerd Dinnebier ◽  
Werner Jaross
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acids ◽  
Nested Pcr ◽  
In Situ Hybridisation ◽  
Target Sequence ◽  
Chain Reaction ◽  
Multiple Control ◽  
Nested Polymerase Chain Reaction ◽  
Polymerase Chain

In the present study a protocol of in situ reverse transcriptase‐nested polymerase chain reaction (in situ RT‐nested PCR) was examined based on the following modifications. (i) To exclude false positive signals caused by “DNA repair mechanisms” and “endogenous priming”, a two‐step PCR was applied after reverse transcription. The first step was performed in the presence of extrinsic primers and unlabeled nucleotides with a maximum of PCR cycles possible without destroying the cell morphology. The second step consisted of only one annealing/elongation reaction, the target sequence marked by addition of digoxigenin‐labeled nucleotides and intrinsic primers. (ii) In order to prevent amplifications of genomic DNA nested primer pairs were applied crossing intron sequences. (iii) To minimize the diffusion of PCR products in cells, the extrinsic primers were extended with complementary 5′‐tails. This approach results in the generation of high molecular weight concatamers during PCR cycles. By applying this protocol, immunostainings specific for phospholipase A2 of type IIA mRNA were exclusively detectable in the cytoplasm of HepG2 hepatoma cells, which were used as a model system, whereas the nuclei were unstained. Multiple control experiments yielded completely negative results. These data suggest that the in situ RT‐nested PCR, which in comparison to the method of in situ RT‐PCR‐in situ‐hybridisation is simpler and less time‐consuming, can be used as an alternative approach to identify intracellular nucleic acids.

Application of Nested Polymerase Chain Reaction to Detection of Salmonella in Poultry Environment

2002 ◽  
Vol 65 (8) ◽  
pp. 1227-1232 ◽  
Author(s):  
TONGRUI LIU ◽  
KAREN LILJEBJELKE ◽  
ELIZABETH BARTLETT ◽  
CHARLES HOFACRE ◽  
SUSAN SANCHEZ ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Nested Pcr ◽  
Virulence Gene ◽  
Pcr Primers ◽  
Processing Plant ◽  
Chain Reaction ◽  
Chi Square ◽  
Nested Polymerase Chain Reaction ◽  
Secondary Enrichment ◽  
Polymerase Chain

Isolation of Salmonella from environmental and processing-plant poultry samples requires the sampling of large numbers of areas within the poultry house or plant. Subsequently, the required number of samples necessitates a large volume of work for a microbiology laboratory, especially when the protocol requires the inclusion of a delayed secondary enrichment for the isolation of Salmonella. This study examined the use of the polymerase chain reaction (PCR) to identify those secondary enrichments containing Salmonella. The unique Salmonella virulence gene invA was chosen as the target for the development of a nested PCR because of its uniform distribution among Salmonella serotypes. The use of nested PCR primers increased the sensitivity of detection 100-fold, resulting in the detection of as few as four cells. There was a strong, statistically significant positive correlation between PCR and culture results as determined by chi-square (P < 0.001) and kappa (k = 0.915; excellent agreement) tests. Using PCR to screen primary enrichments for presumptive Salmonella contamination, we improved our efficiency at isolating Salmonella upon secondary enrichment by 20%, and no false negatives were observed. This method will not only validate the use of secondary enrichment procedures but also reduce costs and manpower required for the surveillance of Salmonella.

scholarly journals Prediction of Clostridium difficile infection in patients with polymerase chain reaction using a classification and regression tree

2020 ◽  
Author(s):  
Diana Martínez-Ruíz ◽  
Luis Gabriel Parra-Lara ◽  
Fernando Rosso
Keyword(s):  
Polymerase Chain Reaction ◽  
Clostridium Difficile ◽  
Negative Predictive Value ◽  
Predictive Value ◽  
Regression Tree ◽  
Antifungal Drugs ◽  
Classification And Regression Tree ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Classification And Regression

Abstract BackgroundClostridium difficile infection (CDI) is commonly diagnosed with the polymerase chain reaction (PCR), but this test finds a high percentage of false positives, so their use and interpretation in CDI is a challenge in the clinical practice. That is why it is necessary to define an algorithm to optimize the use of PCR that considers clinical characteristics to classify patients with diarrhea as CDI or without CDI. ObjectiveTo identify a predictive algorithm with the clinical features that best classify patients with CDI vs. without CDI, to help physicians in making decisions to request PCR. Materials and methodsA case-control study was conducted at Fundación Valle del Lili. The population was inpatients between 2012 and 2016, with 18 or more years, and diarrhea, abdominal pain, or other nonspecific gastrointestinal symptoms who underwent PCR. Cases were defined as patients with positive PCR for C. difficile and as controls patients with negative PCR for C. difficile. Predictive algorithms to classify patients was constructed using a classification tree, classification and regression tree (CART). ResultsA total of 149 patients were included (48 cases with positive PCR and 99 controls with negative PCR). The CART has a high capacity to classify patients with a negative PCR correctly. It includes variables about the history of antibiotics use, the use of proton-pump inhibitor, the use of ranitidine, and the use of antifungal drugs. The CART showed sensitivity 64.6%, specificity 85.8%, positive predictive value 68.8%, negative predictive value 83.3%. and AUC 79.7%.DiscussionCART had good specificity and a high negative predictive value; it could be considered as an algorithm to identify conditions that indicate when it is not necessary to perform a PCR test in a patient symptom of CDI.

scholarly journals Epidemiology of epizootic lymphangitis of carthorses in northern Ethiopia using conventional diagnostic methods and nested polymerase chain reaction

2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Birhanu Hadush ◽  
Molla Michaelay ◽  
Habtamu Taddele Menghistu ◽  
Nigus Abebe ◽  
Abreha Tesfaye Genzebu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Clinical Examination ◽  
Nested Pcr ◽  
Control Measure ◽  
Genetic Material ◽  
Diagnostic Methods ◽  
Northern Ethiopia ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Polymerase Chain

Abstract Background Epizootic lymphangitis (EL), caused by Histoplasma capsulatum variety farciminosum (HCF) is a contagious, chronic disease of equines, characterized by development of nodular lesions in the lymph nodes, lymphatic vessels and skin. It is one of the most important diseases of equines in Ethiopia, causing significant economic loss, particularly in the livelihood of carthorse owners. To date there is neither effective diagnostic nor control measure implemented in the country. Furthermore, there is a shortage of data on the epidemiology of the disease in different regions of this country. The aim of this study was to investigate epidemiology of EL in northern Ethiopia, using the conventional methods as well as nested polymerase chain reaction (PCR). Results The presence of HCF genetic material was confirmed in 44% (84/191) of the carthorses. Subclinical infection was observed in 18.2% (22/121) of the apparently healthy carthorses. Considering the nested PCR as a gold standard, sensitivity and specificity of clinical examination were 74% and 92.5%, respectively, while the area under the ROC curve (AUR) was 0.83 (95% confidence interval, 0.77, 0.896). Moreover, a moderate (k = 0.675) agreement observed between the nested PCR and clinical examination. Conclusions This study demonstrated widespread occurrence of EL in northern Ethiopia, and the advantage of the nested PCR in detecting infection of HCF, even before the clinical symptoms became apparent.

scholarly journals Performance of a Multiplex Nested Polymerase Chain Reaction in Detecting 7 Pathogens Containing DNA in Their Genomes Associated With Congenital Infections

2019 ◽  
Vol 144 (1) ◽  
pp. 99-106
Author(s):  
Lidia Yamamoto ◽  
Antonio G. Amorim Filho ◽  
Joelma A. Queiroz ◽  
Mario H. B. de Carvalho ◽  
Jonatas C. Rodrigues ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Nested Pcr ◽  
Detection Rate ◽  
Simultaneous Detection ◽  
Immunoglobulin M ◽  
Fisher Exact Test ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Congenital Infections ◽  
Polymerase Chain

Context.— Infections are the leading cause of perinatal and infant mortality in low-income and low-resource countries, which have a higher prevalence of infections. Definitive diagnosis of congenital and perinatal infections is largely dependent upon the results of laboratory tests. Objective.— To develop a multiplex nested polymerase chain reaction (PCR) technique for the simultaneous detection of 7 pathogens containing DNA in their genomes in suspected cases of congenital infection. Design.— Eligible participants were pregnant women with positive immunoglobulin M antibodies raised to one of the pathogens in the prenatal serologic screening, associated or not with fetal ultrasound abnormalities or positive fetal serology. Neonates whose mothers did not attend prenatal care were included when they presented with symptomatology and laboratory parameters suggestive of infection. The detection rate of the multiplex nested PCR was compared with maternal, fetal, and neonatal serology, as well as placental immunohistochemistry and noncommercial amplifications. Results.— Of 161 suspected cases, the multiplex nested PCR detected 60 (37.3%), whereas the tests available in hospital laboratories detected 13 of 60 (21.7%) of the cases detected by the multiplex nested PCR, demonstrating a 4.6 times higher detection rate for the multiplex nested PCR (Fisher exact test, P < .001). Positive amplifications were to Toxoplasma gondii (32 cases), cytomegalovirus (14 cases), parvovirus B19 (5 cases), and adenovirus (5 cases). In 4 cases, 2 pathogens were simultaneously detected. All types of biological matrices were suitable for amplification. Sequencing of multiplex nested PCR products confirmed the molecular findings. Conclusions.— The multiplex nested PCR significantly increased the number of diagnosed congenital infections. Given the scarcity of DNA recovered from amniotic fluid and some neonatal samples, this multiplex nested PCR allows the simultaneous detection of 7 pathogens associated with congenital infections in a reliable, faster, cost-effective, and more sensitive way.

scholarly journals Development of a Specific Polymerase Chain Reaction System for the Detection of Rice Orange Leaf Phytoplasma

Plant Disease ◽  
2020 ◽  
Vol 104 (2) ◽  
pp. 521-526
Author(s):  
Zhiyi Wang ◽  
Yingzhi Zhu ◽  
Zhanbiao Li ◽  
Xin Yang ◽  
Tong Zhang ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Nested Pcr ◽  
False Positive ◽  
Southern China ◽  
Reaction System ◽  
Universal Primers ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Orange Leaf ◽  
Polymerase Chain

Rice orange leaf disease (ROLD), caused by rice orange leaf phytoplasma (ROLP), is transmitted by leafhopper vectors Recilia dorsalis and Nephotettix cinticeps. ROLD severely devastates rice production in Asia. Accurate detection of the pathogen is important for disease management. Current nested polymerase chain reaction (nested PCR) method using phytoplasma universal primers is widely used to detect phytoplasmas; however, it has shortcoming of inconvenience and inaccuracy, for it needs two round of PCR reactions and could produce false positive results due to nontarget amplification. In this study, we developed a PCR assay using a set of primers designed based on the ROLP genome sequence to amplify house-keeping gene FtsH-1 in rice and leafhopper vector samples. This method is simple and rapid, and its sensitivity up to 10 pg/μl of total ROLP DNA. It also minimizes the false positive problem produced by nested PCR. This method was used to survey the geographic distribution of ROLD in southern China from 2016 to 2018. The results showed that the distribution areas and vector carrying rate of ROLD had gradually increased.

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