Nested polymerase chain reaction for detection of pathogenic leptospires

Leptospirosis is a widespread zoonosis caused by pathogenic members of the genus Leptospira that has a great impact on human and veterinary public health. Early diagnosis of leptospirosis is important because severe lepto spiral infection can have a fulminant course. The available serological techniques for the diagnosis of leptospirosis have low sensitivity during the early stage of the disease. Efforts are being made to develop simpler, effective, efficient, and inexpensive diagnostic methods. In this work, we first evaluate a polymerase chain reaction (PCR) based method for diagnosis of leptospirosis. Primers were designed to amplify a 264 bp region within the lipL32 gene that is conserved among pathogenic Leptospira and absent in nonpathogenic species. The sensitivity and specificity of the assay were evaluated using 7 saprophytic serovars, 37 pathogenic serovars, and 15 other microorganisms. The method was very specific for pathogenic serovars, however, it lacked sensitivity. To enhance the sensitivity, another primer pair was designed to amplify a 183 bp region within the 264 bp region of the lipL32 gene and was used in a nested PCR assay. This approach was much more sensitive than conventional PCR.Key words: leptospirosis, diagnosis, nested PCR, lipL32.

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

10.21203/rs.2.19415/v1 ◽

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.

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

BMC Veterinary Research ◽

10.1186/s12917-020-02582-2 ◽

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.

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

Analytical Cellular Pathology ◽

10.1155/2001/654016 ◽

2001 ◽

Vol 22 (3) ◽

pp. 151-158 ◽

Cited By ~ 1

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.

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

Journal of Food Protection ◽

10.4315/0362-028x-65.8.1227 ◽

2002 ◽

Vol 65 (8) ◽

pp. 1227-1232 ◽

Cited By ~ 36

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.

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Corrigendum to “Giardia and Cryptosporidium spp. dissemination during wastewatertreatment and comparative detection via immunofluorescence assay (IFA), nested polymerase chain reaction (nested PCR) and loopmediated isothermal amplification (LAMP)” [Acta Trop. 158 (2016) 43–51]

Acta Tropica ◽

10.1016/j.actatropica.2016.10.026 ◽

2017 ◽

Vol 166 ◽

pp. 363 ◽

Cited By ~ 1

Author(s):

Carmen Gallas-Lindemann ◽

Isaia Sotiriadou ◽

Judit Plutzer ◽

Michael J. Noack ◽

Mohammad Reza Mahmoudi ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Nested Pcr ◽

Immunofluorescence Assay ◽

Isothermal Amplification ◽

Chain Reaction ◽

Nested Polymerase Chain Reaction ◽

Cryptosporidium Spp ◽

Polymerase Chain

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Low Sensitivity of a Nested Polymerase Chain Reaction in Oropharyngeal Washings for the Diagnosis of Pneumocystis Pneumonia in HIV-Infected Patients

CHEST Journal ◽

10.1378/chest.128.1.167 ◽

2005 ◽

Vol 128 (1) ◽

pp. 167-171 ◽

Cited By ~ 8

Author(s):

Kennedy Nyamande ◽

Umesh G. Lalloo ◽

Dennis York ◽

Mogambal Naidoo ◽

Elvis M. Irusen ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Pneumocystis Pneumonia ◽

Chain Reaction ◽

Nested Polymerase Chain Reaction ◽

Polymerase Chain ◽

Low Sensitivity

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Polymerase Chain Reaction-Assisted Evaluation of the Efficacy of Seed-Treatment Prevention of Sporisorium reilianum Infection in Sorghum Seedlings

Frontiers in Microbiology ◽

10.3389/fmicb.2021.745144 ◽

2021 ◽

Vol 12 ◽

Author(s):

Zhi Zhang ◽

Juan Fan ◽

Mucai Feng ◽

Hongbo Qiu ◽

Anlong Hu

Keyword(s):

Polymerase Chain Reaction ◽

Seed Treatment ◽

Early Stage ◽

Disease Incidence ◽

Infected Plant ◽

Seed Treatments ◽

Chain Reaction ◽

Pcr Assay ◽

Sporisorium Reilianum ◽

Polymerase Chain

Head smut, caused by Sporisorium reilianum [(Kuhn) Langdon and Fullerton], is a major disease of sorghum. Seed treatment is considered to be the most effective way to control the disease; however, the pathogen can infect at the seedling stage and the infected plant will not display symptoms until the reproductive stage is reached. The evaluation of the efficacy of seed treatments is time consuming and is dependent upon visible symptoms. Polymerase chain reaction (PCR) methods have the ability to identify pathogens and diagnose their presence at an early stage of infection. In this study, the S. reilianum-specific primer SR3 was used for PCR detection pathogen. We optimized temperature, humidity, and spore quantity test conditions and were able to achieve >88% infection incidence in sorghum seedlings. Sorghum seeds were soaked in various concentrations of tebuconazole and planted for 7 days in soil containing 0.2% teliospores. The efficacy of tebuconazole against S. reilianum was evaluated by PCR and recorded as disease incidence. Results indicated that the reduction in disease incidence after exposure to 0.15, 0.30, 0.45, 0.60, and 0.75 μg/mL tebuconazole was 6.24, 37.48, 67.74, 81.24, and 93.74%, respectively. Significant differences between the concentrations of tebuconazole were observed. The PCR assay represents a valuable tool for evaluating the efficacy of fungicide seed treatments for the control of S. reilianum in sorghum under laboratory conditions.

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

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2018-0544-oa ◽

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.

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

Plant Disease ◽

10.1094/pdis-05-19-1047-re ◽

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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