Molecular Biology for the Clinician: Understanding Current Methods

2006 ◽  
Vol 42 (5) ◽  
pp. 326-335 ◽  
Author(s):  
Heather L. Chandler ◽  
Carmen M.H. Colitz
Keyword(s):  
Molecular Biology ◽  
Blot Analysis ◽  
Deoxyribonucleic Acid ◽  
Northern Blot Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Biochemical Assays ◽  
Basic Understanding ◽  
Polymerase Chain ◽  
Pcr Microarray

The basics of molecular biology involve the replication of deoxyribonucleic acid and its transcription and translation into proteins. Biochemical assays such as the Southern blot analysis, polymerase chain reaction (PCR), Northern blot analysis, reverse-transcriptase PCR, microarray technology, Western blot analysis, immunohistochemistry, enzyme-linked immunosorbent assay, and flow cytometry utilize various aspects of molecular biology. To understand these assays requires some basic understanding of the principles of molecular biology. This paper provides basic information on the methodology and techniques used in these assays.

scholarly journals Tobacco Plants Transformed with a Modified Coat Protein of Tomato Mottle Begomovirus Show Resistance to Virus Infection

1999 ◽  
Vol 89 (8) ◽  
pp. 701-706 ◽  
Author(s):  
X. H. Sinisterra ◽  
J. E. Polston ◽  
A. M. Abouzid ◽  
E. Hiebert
Keyword(s):  
Coat Protein ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Protein Gene ◽  
Binary Vector ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Resistance Response ◽  
Tobacco Plants ◽  
Polymerase Chain

Tobacco plants (Nicotiana tabacum ‘Xanthi’) were transformed with a binary vector containing the coat protein gene of tomato mottle begomo-virus (ToMoV) modified by the deletion of 30 nucleotides in the 5′ end. The R1 generation was screened for resistance to ToMoV by inoculation with viruliferous whiteflies. Fifteen days after inoculation, symptom development was recorded weekly for up to 120 days using a visual scale, and ToMoV infection was confirmed by polymerase chain reaction and enzyme-linked immunosorbent assay. The response to high inoculation levels of ToMoV varied and ranged from susceptibility to immunity. The transgene transcript was detected by northern blot analysis; however, the transgene product could not be detected by protein blot analysis using antisera reactive with ToMoV coat protein. The lack of detection of the transgene product in resistant plants suggests that it is not involved in eliciting the resistance response and that resistance may be mediated by the transgene transcript.

Cloning and widespread distribution of the rat rod-type cyclic nucleotide-gated cation channel

1997 ◽  
Vol 272 (4) ◽  
pp. C1335-C1344 ◽  
Author(s):  
C. Ding ◽  
E. D. Potter ◽  
W. Qiu ◽  
S. L. Coon ◽  
M. A. Levine ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
In Situ Hybridization ◽  
Pineal Gland ◽  
Northern Blot ◽  
Blot Analysis ◽  
Cyclic Nucleotide ◽  
Northern Blot Analysis ◽  
Chain Reaction ◽  
Polymerase Chain

We used Northern blot analysis, ribonuclease protection assay (RPA), reverse transcriptase-polymerase chain reaction, and in situ hybridization to investigate the hypothesis that the CNG1 isoform of the cyclic nucleotide-gated nonselective cation channel may be widely distributed in tissues of the rat. A cDNA encoding the CNG1 isoform was isolated from rat eye and human retina, and partial sequences were isolated from rat pineal gland and human kidney. Northern blot analysis revealed a 3.1-kilobase (kb) CNG1 transcript in rat eye, pineal gland, pituitary, adrenal gland, and spleen, and a larger transcript of 3.5 kb was found in testis. RPA confirmed the identity of CNG1 mRNA in rat eye, lung, spleen, and brain. Polymerase chain reaction-based detection of the mRNA for CNG1 indicates that the channel is expressed in lower abundance in many other tissues, including thymus, skeletal muscle, heart, and parathyroid gland. The cellular distribution of CNG1 was further studied by in situ hybridization, which demonstrated expression of mRNA in lung, thymus, pineal gland, hippocampus, cerebellum, and cerebral cortex but not in heart or kidney.

Clinical Relevance of Annual Screening Using a Commercial Enzyme-Linked Immunosorbent Assay (SNAP 3Dx) for Canine Ehrlichiosis

2009 ◽  
Vol 45 (3) ◽  
pp. 118-124 ◽  
Author(s):  
Barbara C. Hegarty ◽  
Pedro Paulo Vissotto de Paiva Diniz ◽  
Julie M. Bradley ◽  
Leif Lorentzen ◽  
Edward Breitschwerdt
Keyword(s):  
Clinical Relevance ◽  
Deoxyribonucleic Acid ◽  
Immunofluorescence Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Active Infection ◽  
Chain Reaction ◽  
Canine Ehrlichiosis ◽  
Annual Screening ◽  
Polymerase Chain ◽  
Positive Results

Eighty-six dogs were selected based upon Ehrlichia (E.) canis SNAP 3Dx positive results to determine clinical relevance of annual E. canis screening. Immunofluorescence assay showed 72 (84%) of 86 dogs were seroreactive for E. canis. Polymerase chain reaction (PCR) revealed that 12 (14%) of 86 dogs had Ehrlichia deoxyribonucleic acid; seven had E. canis, four had E. ewingii, and one was coinfected with E. chaffeensis and E. ewingii. Thrombocytopenia (<164,000 platelets/μL) was found in 28 (39%) of 72 dogs. In this study, thrombocytopenia was frequently detected in healthy Ehrlichia SNAP 3Dx-positive dogs, whereas active infection was infrequently confirmed by PCR. Therefore, treatment based upon screening results alone is not recommended.

scholarly journals Genotyping ofSalmonellaspp. on the basis of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

2018 ◽  
Author(s):  
Calvin Jiksing ◽  
Normah Yusop ◽  
Farhan Nazaie Nasib ◽  
Kenneth Francis Rodrigues
Keyword(s):  
Polymerase Chain Reaction ◽  
Mycobacterium Tuberculosis ◽  
Immunosorbent Assay ◽  
Deoxyribonucleic Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Pcr Polymerase Chain Reaction ◽  
Crispr Array ◽  
Polymerase Chain ◽  
Veterinary Laboratory

ABSTRACTAimsBacterial genotyping on the basis of the CRISPR array has been established inMycobacterium tuberculosiswith a method called spacer oligonucleotide typing (spoligotyping). The spoligotyping method had been widely used for both detection and typing ofM. tuberculosiscomplex bacteria. This present study aimed at determining if the CRISPR array inSalmonellaspp. could be applied to establish a correlationship between serogroup and the fingerprint generated by CRISPR typing.Methodology and resultsA total of 30 samples were obtained from Diagnostic Veterinary Laboratory, Kota Kinabalu, Sabah. Serogroup was determined on the basis of ELISA (enzyme-linked immunosorbent assay). Four different serogroups were identified which were serogroup B, C, D, and E. DNA (deoxyribonucleic acid) was extracted and PCR (polymerase chain reaction) was performed using primers which were designed to amplify the CRISPR array inSalmonellagenome. Our results indicate that there is a correlationship between serogroup obtained using ELISA and the profile generated by CRISPR typing.Conclusion, significance and impact of studyCRISPR typing has the potential to be applied for the genotyping ofSalmonella.

scholarly journals Evaluation of Sandwich Enzyme-Linked Immunosorbent Assay and Reverse Transcription Polymerase Chain Reaction for the Diagnosis of Foot-and-Mouth Disease

Intervirology ◽  
2021 ◽  
pp. 1-6
Author(s):  
Salman Khan ◽  
Syed Asad Ali Shah ◽  
Syed Muhammad Jamal
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Genome ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Kappa Value ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Level Of Agreement

<b><i>Background:</i></b> Foot-and-mouth disease (FMD) is an infectious and highly contagious disease of cloven-hoofed domestic and wild animals, causing heavy economic losses to the livestock industry. Rapid and reliable diagnosis of the disease is essential for the implementation of effective control measures. This study compared sandwich enzyme-linked immunosorbent assay (S-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of FMD. <b><i>Methods:</i></b> A total of 60 epithelial samples from suspected cases of FMD were tested using both S-ELISA and RT-PCR assays. The level of agreement between the assays was assessed by calculating the Kappa value. <b><i>Results:</i></b> S-ELISA detected 38 (63%) samples positive for FMD virus (FMDV). Being predominant, serotype O was detected in 22 (57.9%) of the total samples tested positive, whereas 9 (23.7%) and 7 (18.4%) samples were found positive for serotypes A and Asia-1, respectively. RT-PCR detected viral genome in 51 (85%) of the samples using pan-FMDV primers set, 1F/1R. Thirty-six samples were found positive and 7 negative by both the tests. The level of agreement between the tests was assessed by calculating the Kappa value, which was found to be fair (Kappa value = 0.303 and 95% CI = 0.089; 0.517) and significant (<i>p</i> = 0.009). However, 2 samples, which were found positive on S-ELISA tested negative on RT-PCR. This may be attributed to the presence of nucleotide mismatch(es) in the primer-binding sites that may have resulted in failure of amplification of the viral genome. The serotype-specific RT-PCR assays not only confirmed serotyping results of S-ELISA but were also able to establish serotype in 9 S-ELISA-negative but pan-FMDV RT-PCR-positive samples. <b><i>Conclusions:</i></b> The RT-PCR assay contributes significantly to establishing a quick, sensitive, and definitive diagnosis of FMD in resource-constrained countries. Samples giving negative results in S-ELISA should be tested in RT-PCR for the disease detection and virus typing.

scholarly journals Occurrence of abortions induced by Neospora caninum in dairy cattle from Santa Catarina, southern Brazil

2017 ◽  
Vol 26 (3) ◽  
pp. 292-298 ◽  
Author(s):  
Cesar Augusto Barbosa de Macedo ◽  
Madlaine Frigo Silveira Barbosa de Macedo ◽  
Ana Carolina Miura ◽  
Alessandra Taroda ◽  
Sergio Tosi Cardim ◽  
...  
Keyword(s):  
Dairy Cattle ◽  
Dairy Cows ◽  
High Frequency ◽  
Neospora Caninum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Southern Brazil ◽  
Chain Reaction ◽  
Santa Catarina ◽  
Tissue Samples ◽  
Polymerase Chain

Abstract The aim of the present study was to investigate the occurrence of N. caninum associated with abortions of dairy cattle from Santa Catarina state, southern Brazil by using enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and polymerase chain reaction (PCR). Blood from dairy cows that aborted along with intrathoracic fluid and tissue samples (brain, heart, liver, and lung) from their fetuses were collected and used for serology; PCR, histopathological, and immunohistochemistry (IHC) evaluations were also conducted. Twenty-one cows (51.2%) out of 41, and eight fetuses (26.7%) out of 30 were ELISA (HerdCheck, IDEXX) positive for N. caninum. Dams > 36 months of age had a higher risk of being serum positive than younger animals. PCR and IHC revealed that 38.8% (14/36) and 25.0% (9/36) of the fetuses were positive for N. caninum, respectively for each of the tests. Seropositive cows had a higher frequency of fetuses that were also positive by either intrathoracic fluid, PCR, or IHC. In summary, the present study observed a high frequency of N. caninum in abortions from dairy cows from southern Brazil, with a higher N. caninum prevalence found in cows that were older than 36 months. In addition, serology, PCR, and IHC should be used all together for better diagnosis of neosporosis in cattle.

scholarly journals Bartonella spp. in human and animal populations in Gauteng, South Africa, from 2007 to 2009

2012 ◽  
Vol 79 (2) ◽  
Author(s):  
Anastasia N. Trataris ◽  
Jennifer Rossouw ◽  
Lorraine Arntzen ◽  
Allan Karstaedt ◽  
John Frean
Keyword(s):  
Health Care Professionals ◽  
Deoxyribonucleic Acid ◽  
Bartonella Henselae ◽  
Hiv Positive ◽  
Host Immune System ◽  
Wild Rodent ◽  
Chain Reaction ◽  
Blood Samples ◽  
Animal Populations ◽  
Polymerase Chain

Bartonellae are highly adaptive organisms that have the ability to evade the host immune system and cause persistent bacteraemia by occupying the host’s erythrocytes. Bartonella spp. is under-studied and health care professionals often misdiagnose Bartonella-related infections. The aim of this study was to investigate the carriage of Bartonella spp. circulating in human and animal populations in Gauteng using culturing and polymerase chain reaction (PCR) detection. A total of 424 human, 98 cat, 179 dog, and 124 wild rodent blood samples were plated onto specialised media and incubated for 7–21 days at 37 ºC in CO2. Culture isolates morphologically similar to Bartonella control strains were confirmed by PCR and sequenced to determine species. Deoxyribonucleic acid (DNA) was extracted from all blood samples and tested by nested PCR. Bartonella could only be cultured from the cat and rodent specimens. Cat isolates were > 99% similar to Bartonella henselae URBHLIE 9, previously isolated from an endocarditis patient, and rat isolates were > 98% similar to either RN24BJ (candidus ‘Bartonella thailandensis’) or RN28BJ, previously isolated from rodents in China. The PCR prevalences were 22.5% in HIV-positive patients, 9.5% in clinically healthy volunteers, 23.5% in cats, 9% in dogs and 25% in rodents. Findings of this study have important implications for HIV-positive patients.

scholarly journals Comparative Study of Immune-Diagnostic Tools with Polymerase Chain Reaction in Sub-Clinical Leishmaniasis Isolates

2011 ◽  
Vol 12 (1) ◽  
pp. 34-39 ◽  
Author(s):  
Nazar M Abdalla
Keyword(s):  
Polymerase Chain Reaction ◽  
Skin Test ◽  
Diagnostic Methods ◽  
Diagnostic Tools ◽  
Enzyme Linked Immunosorbent Assay ◽  
Clinical Form ◽  
Chain Reaction ◽  
Leishmanin Skin Test ◽  
Wide Range ◽  
Polymerase Chain

Objective: This study aimed to identify cases of leishmaniasis in the Nuba Mountain area, which is situated in a unique geographical site located in the centre of Sudanese leishmania belt. Wide range of investigations are available for detection of leishmania cases, but still the most reliable and easy test used as screening and epidemiological tool in field studies needs to be evaluated. The most commonly used conventional diagnostic methods direct microscopy and culture have some drawbacks in diagnosing subclinical cases of leishmaniasis. Materials and methods: In this study, comparative properties of various immune-diagnostic tools with Polymerase Chain Reaction used in sub-clinical leishmaniasis isolates were explored. The immune-diagnostic tools involved in this study include- Leishmanin Skin Test (LST), Enzyme Linked Immunosorbent Assay (ELISA) and Direct Agglutination Test (DAT). The study was conducted in the Green Valley village (Rashad Province, South Kordofan State) with a population of 332. Most of the villagers presented with sub-clinical form of leishmaniasis with minor symptoms and signs without the features found in clinical form of visceral leishmaniasis such as fever, diarrhoea, epistaxis, enlarged lymph nodes, spleen and liver. In this study we collected demographic, clinical and epidemiological data using special questionnaire. Leishmanin skin test (LST), ELISA, DAT and PCR for parasite DNA detection were used. Result: The final positive cases detected by PCR were 32 out of 332 belong to L. donovani species. The final positive cases detected by LST were 51.2% of the total population under study, while 11 out of the 37 tested samples were positive by ELISA. All of the 332 villagers showed negative readings by DAT with exception of three individuals who were positive with very high titers. Conclusion: DNA etxtraction and amplification with primers can be a good screening tool in subclinical leishmaniasis isolates. Keyword: Sub-clinical; Leishmaniasis; Leishmanin Skin Test; ELISA; DAT; PCR. DOI: 10.3329/jom.v12i1.5422J Medicine 2011; 12 : 34-39

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