scholarly journals Potential Application of PCR Based Molecular Methods in Fish Pathogen Identification: A Review

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Md. Ali Arman Ador ◽  
Md. Shameul Haque ◽  
Sulav Indra Paul ◽  
Jui Chakma ◽  
Rakib Ehsan ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Molecular Techniques ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Resistant Bacteria ◽  
Fish Disease ◽  
Fish Pathogen ◽  
Pathogen Identification ◽  
Chain Reaction ◽  
Polymerase Chain

Molecular biology developments have led to fast growth in new methods for fish disease diagnosis. Molecular diagnostic methods are rapid and more specific, more sensitive than the culture of pathogens, serology, histology, and biochemical methods which are traditionally utilized to identify causative agent fish disease. Molecular diagnostic methods are valuable for detecting specific pathogens that are difficult to culture in vitro or require a long cultivation period and it significantly more rapid in providing results compared to culture. It enables earlier informed decision-making and rapid diagnosis of bacteremia, particularly for low levels of bacteria in specimens. Molecular techniques which have the major significance are mainly PCR-based molecular diagnostic methods including Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Multiplex Polymerase Chain Reaction (multiplexPCR), and Random Amplified Polymorphic DNA (RAPD). These have been increasingly utilized to diagnose fish disease for the last recent years. Molecular diagnostic methods can detect pathogens from asymptomatic fish, so disease outbreaks could be prevented. As a consequence, antibiotic treatment can be reduced and the development of antibiotic-resistant bacteria can be eliminated. In this review paper, we attempt to summarize the potentiality of PCR-based molecular diagnostic methods and their application in fish pathogen identification.

scholarly journals Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

2014 ◽  
Vol 34 (1) ◽  
pp. 29-33 ◽  
Author(s):  
Gisele M. Bacanelli ◽  
Carlos A. N. Ramos ◽  
Flábio R. Araújo
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Anaplasma Marginale ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Chain Reaction ◽  
The Real ◽  
End Point ◽  
Polymerase Chain

The rickettsia Anaplasma marginale is considered the main agent of bovine anaplasmosis. Due the nonspecific clinical signs of the anaplasmosis, the diagnosis of infection depends of laboratory confirmation. In recent years, molecular diagnostic methods have been used to detect A. marginale in cattle. However, the existence of a large number of assays of different sensitivity and cost makes the choice of an appropriate test difficult. In the present study, a real-time Polymerase Chain Reaction (PCR) based on the msp5 target gene was quantitatively assessed and compared to an end point PCR. Both reactions were subjected to sensitivity and specificity evaluation using plasmid DNA and samples from cattle experimentally infected with A. marginale. A comparative field trial of the tests was carried out using samples of cattle from a stable enzootic area for A. marginale. The real-time PCR showed a higher sensitivity than the end point PCR. This reaction (i.e. real-time PCR) was able to detect one copy of the msp5 gene in 100 ηg of plasmidial DNA, and more than 80% of its results were positive among experimentally infected animals seven days after infection. In addition, based on in silico analysis, the real-time PCR evaluated in the present study appears to be useful for the detection of A. ovis.

scholarly journals Nucleic Acid Testing of SARS-CoV-2

2021 ◽  
Vol 22 (11) ◽  
pp. 6150
Author(s):  
Hee-Min Yoo ◽  
Il-Hwan Kim ◽  
Seil Kim
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Reverse Transcription ◽  
Diagnostic Methods ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Nucleic Acid Testing ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

The coronavirus disease 2019 (COVID-19) has caused a large global outbreak. It is accordingly important to develop accurate and rapid diagnostic methods. The polymerase chain reaction (PCR)-based method including reverse transcription-polymerase chain reaction (RT-PCR) is the most widely used assay for the detection of SARS-CoV-2 RNA. Along with the RT-PCR method, digital PCR has emerged as a powerful tool to quantify nucleic acid of the virus with high accuracy and sensitivity. Non-PCR based techniques such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) are considered to be rapid and simple nucleic acid detection methods and were reviewed in this paper. Non-conventional molecular diagnostic methods including next-generation sequencing (NGS), CRISPR-based assays and nanotechnology are improving the accuracy and sensitivity of COVID-19 diagnosis. In this review, we also focus on standardization of SARS-CoV-2 nucleic acid testing and the activity of the National Metrology Institutes (NMIs) and highlight resources such as reference materials (RM) that provide the values of specified properties. Finally, we summarize the useful resources for convenient COVID-19 molecular diagnostics.

scholarly journals Development of PCR Primers to Identify Fusarium oxysporum f. sp. fragariae

Plant Disease ◽  
2013 ◽  
Vol 97 (5) ◽  
pp. 619-625 ◽  
Author(s):  
H. Suga ◽  
Y. Hirayama ◽  
M. Morishima ◽  
T. Suzuki ◽  
K. Kageyama ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Transposable Elements ◽  
Fusarium Oxysporum ◽  
Fungal Pathogen ◽  
Pcr Primers ◽  
Genomic Region ◽  
Molecular Diagnostic ◽  
Pathogen Identification ◽  
Chain Reaction ◽  
Polymerase Chain

Fusarium oxysporum f. sp. fragariae is a fungal pathogen causing Fusarium wilt on strawberry. Polymerase chain reaction (PCR) primers that can discriminate F. oxysporum f. sp. fragariae from nonpathogenic F. oxysporum would greatly assist pathogen identification. In order to develop a molecular diagnostic tool for this pathogen, transposable elements in the pathogen were characterized and used for designing a specific set of PCR primers. Portions of the transposable elements Fot3, Han, Hop, Hornet1, and Skippy were detected in all 33 strains of F. oxysporum f. sp. fragariae tested by PCR, whereas Foxy was detected in 32 strains and Impala sequences were detected in 30 strains. Two types of sequences were detected for Hop, two types for Impala, and three types for Skippy. The genomic region between Han and Skippy was amplified by an inter-retrotransposon amplified polymorphism technique, and PCR primers (FofraF and FofraR) to specifically identify F. oxysporum f. sp. fragariae were designed from this region. The developed PCR primers discriminated F. oxysporum f. sp. fragariae strains from nonpathogenic F. oxysporum strains and five other formae speciales. Conidia of F. oxysporum f. sp. fragariae could be detected in brown lowland-type soil by PCR using the primers. After preculturing the soil sample on FoG2 medium, 1 × 102 conidia/g of soil could be detected; without preculturing, 1 × 103 conidia/g of soil were detected.

Acid-fast Smear and Histopathology Results Provide Guidance for the Appropriate Use of Broad-Range Polymerase Chain Reaction and Sequencing for Mycobacteria

2015 ◽  
Vol 139 (8) ◽  
pp. 1020-1023 ◽  
Author(s):  
Kennon Miller ◽  
Susan M. Harrington ◽  
Gary W. Procop
Keyword(s):  
Polymerase Chain Reaction ◽  
Test Performance ◽  
False Negative ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Chain Reaction ◽  
Mycobacterium Tuberculosis Infection ◽  
Primary Methods ◽  
Clinical Specimens ◽  
Polymerase Chain

Context New molecular diagnostic tests are attractive because of the potential they hold for improving diagnostics in microbiology. The value of these tests, which is often assumed, should be investigated to determine the best use of these potentially powerful tools. Objective To investigate the usefulness of broad-range polymerase chain reaction (PCR), followed by sequencing, in mycobacterial infections. Design We reviewed the test performance of acid-fast bacilli (AFB) PCR and traditional diagnostic methods (histopathology, AFB smear, and culture). We assessed the diagnostic effect and cost of the unrestricted ordering of broad-range PCR for the detection and identification of mycobacteria in clinical specimens. Results The AFB PCR was less sensitive than culture and histopathology and was less specific than culture, AFB smear, and histopathology. During 18 months, $93 063 was spent on 183 patient specimens for broad-range PCR and DNA sequencing for mycobacteria to confirm one culture-proven Mycobacterium tuberculosis infection that was also known to be positive by AFB smear and histopathology. In this cohort, there was a false-negative AFB PCR for M tuberculosis and a false-positive AFB PCR for Mycobacterium lentiflavum. Conclusion Testing of AFB smear–negative specimens from patients without an inflammatory response supportive of a mycobacterial infection is costly and has not been proven to improve patient care. Traditional diagnostics (histopathology, AFB smear, and culture) should remain the primary methods for the detection of mycobacteria in clinical specimens.

scholarly journals Polymerase chain reaction for detection of some highly dangerous viral fish disease agents

2020 ◽  
pp. 11-16
Author(s):  
A. S. Tarasova ◽  
A. V. Perchun ◽  
V. P. Melnikov
Keyword(s):  
Polymerase Chain Reaction ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Analytical Sensitivity ◽  
Chain Reaction ◽  
Fish Diseases ◽  
Viral Hemorrhagic Septicemia ◽  
Infectious Salmon Anemia ◽  
Infectious Hematopoietic Necrosis ◽  
Polymerase Chain

Today viral fish diseases cause major losses in the world aquaculture. Pathogen spread often occurs during the transportation of fish from infected farms to the disease-free ones. Therefore, the import of fish stocking material to Russia from countries with a different epidemic situation requires risk-based monitoring and forecasting. Diagnostics is of primary importance in the complex of measures to prevent the spread of viral infections in fish. To date, laboratory diagnostics of viral fish diseases is based on pathogen isolation and its identification using serological methods which require a lot of time and are performed only in large research institutes with specialized laboratories. Molecular diagnostic methods are more sensitive and high-performance. The article presents the results of using reverse transcription polymerase chain reaction to detect a number of highly dangerous viral diseases of fish (Salmonidae). As a result of this work, primers were selected and the temperature and time conditions of the reaction were optimized for the identification of infectious hematopoietic necrosis, viral hemorrhagic septicemia and infectious salmon anemia. The results obtained during the research allowed us to establish that this diagnostic method is highly specific with analytical sensitivity to infectious salmon anemia virus of 2.5 lg TCD50/сm3, to infectious hematopoietic necrosis – 2.9 lg TCD50/сm3 and to viral hemorrhagic septicemia – 4.2 lg TCD50/ сm3. The described method was used to identify reference and field strains available at the FGBI ARRIAH Reference Laboratory for Aquaculture Diseases and isolated in different years in fish farms in the territory of the Russian Federation. The research data correlated with the results obtained from virus neutralization in cell culture and ELISA performed using commercial kits. The proposed method of RT-PCR allows to detect pathogens both in fish with pronounced clinical signs and in latent virus carriers.

Diagnostic Accuracy of a Direct Panfungal Polymerase Chain Reaction Assay Performed on Stained Cytology Slides

2021 ◽  
pp. 030098582199156
Author(s):  
Alexandra N. Myers ◽  
Unity Jeffery ◽  
Zachary G. Seyler ◽  
Sara D. Lawhon ◽  
Aline Rodrigues Hoffmann
Keyword(s):  
Polymerase Chain Reaction ◽  
Diagnostic Accuracy ◽  
Molecular Techniques ◽  
Fungal Culture ◽  
Chain Reaction ◽  
Identification Of Fungi ◽  
Fungal Dna ◽  
Polymerase Chain ◽  
Panfungal Pcr ◽  
Pcr Targeting

Molecular techniques are increasingly being applied to stained cytology slides for the diagnosis of neoplastic and infectious diseases. Such techniques for the identification of fungi from stained cytology slides have not yet been evaluated. This study aimed to assess the diagnostic accuracy of direct (without nucleic acid isolation) panfungal polymerase chain reaction (PCR) followed by sequencing for identification of fungi and oomycetes on stained cytology slides from dogs, cats, horses, and other species. Thirty-six cases were identified with cytologically identifiable fungi/oomycetes and concurrent identification via fungal culture or immunoassay. Twenty-nine controls were identified with no cytologically or histologically visible organisms and a concurrent negative fungal culture. Direct PCR targeting the internal transcribed spacer region followed by sequencing was performed on one cytology slide from each case and control, and the sensitivity and specificity of the assay were calculated. The sensitivity of the panfungal PCR assay performed on stained cytology slides was 67% overall, 73% excluding cases with oomycetes, and 86% when considering only slides with abundant fungi. The specificity was 62%, which was attributed to amplification of fungal DNA from control slides with no visible fungus and negative culture results. Direct panfungal PCR is capable of providing genus- or species-level identification of fungi from stained cytology slides. Given the potential of panfungal PCR to amplify contaminant fungal DNA, this assay should be performed on slides with visible fungi and interpreted in conjunction with morphologic assessment by a clinical pathologist.

Comparison of Droplet Digital Polymerase Chain Reaction (ddPCR) and Real-Time Quantitative Polymerase Chain Reaction (qPCR) in Detecting Neonatal Invasive Fungal Infections

2021 ◽  
Vol 11 (3) ◽  
pp. 373-379
Author(s):  
Huitao Li ◽  
Xueyu Chen ◽  
Xiaomei Qiu ◽  
Weimin Huang ◽  
Chuanzhong Yang
Keyword(s):  
Polymerase Chain Reaction ◽  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Methods ◽  
Chain Reaction ◽  
Fungal Isolation ◽  
Blood Samples ◽  
Fungal Strains ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Invasive fungal infection (IFI) is the leading cause of death in neonatal patients, yet the diagnosis of IFI remains a major challenge. At present, most IFI laboratory diagnostic methods are based on classical, but limited, methods such as fungal isolation and culture and histopathological examination. Recently, quantitative polymerase chain reaction (qPCR) and droplet digital polymerase chain reaction (ddPCR) technology have been adopted to quantify nucleic-acid identification. In this study, we established qPCR and ddPCR assays for IFI diagnosis and quantification. qPCR and ddPCR were carried out using identical primers and probe for the amplification of 18S rRNA. Assay results for three fungal strains were positive, whereas ten non-fungal strains had negative results, indicating 100% specificity for both ddPCR and qPCR methods. Genomic DNA of Candida albicans was tested after a serial dilution to compare the sensitivity of the two PCR methods. The limit of detection of ddPCR was 3.2 copies/L, which was a ten-fold increase compared with that of the qPCR method (32 copies/L). Blood samples from 127 patients with high-risk factors and clinical symptoms for IFI were collected from a NICU in Shenzhen, China, and analyzed using qPCR and ddPCR. Thirty-four blood samples from neonates had a proven or probable diagnosis of IFI, and 25 of these were positive by qPCR, whereas 30 were positive by ddPCR. Among the 93 blood samples from neonates who had a possible IFI or no IFI, 24 were positive using qPCR, and 7 were positive using ddPCR. In conclusion, ddPCR is a rapid and accurate pan-fungal detection method and provides a promising prospect for IFI clinical screening.

scholarly journals UTILITY OF ANTIGEN DETECTION TEST AND POLYMERASE CHAIN REACTION IN THE DIFFERENTIATION OF TUBERCULOUS AND NON-TUBERCULOUS MYCOBACTERIA

2017 ◽  
Vol 10 (6) ◽  
pp. 289
Author(s):  
Yogita Singh ◽  
Raji Vasanth ◽  
Shrikala Baliga ◽  
Dhanashree B
Keyword(s):  
Polymerase Chain Reaction ◽  
False Negative ◽  
Diagnostic Methods ◽  
Clinical Samples ◽  
Chain Reaction ◽  
College Hospital ◽  
Medical College ◽  
Negative Results ◽  
Polymerase Chain ◽  
Mycobacterium Spp

Objectives: Cultivation and identification of mycobacteria to species level remains difficult and time-consuming. Hence, easy and rapid diagnostic methods are necessary for the differentiation of Mycobacterium tuberculosis (MTB) from non-tuberculous mycobacteria (NTM). The present study aims to detect and differentiate MTB from NTM isolated from clinical samples by immunochromatographic test (ICT) and polymerase chain reaction (PCR). Methods: Over a period of 1 year, clinical samples (n=496) received from suspected cases of TB, at the Department of Microbiology, Kasturba Medical College Hospital, Mangalore were cultured to isolate Mycobacterium spp. Identification of all the isolates was done by conventional biochemical technique, ICT, and PCR. Results: Among the 496 samples processed, 49 (9.87%) were acid-fast bacilli smear positive and 59 (11.89%) samples showed the growth of Mycobacterium spp. Among these, 10 were rapid growers, 49 were slow-growing mycobacteria, out of which 30 were MTB as identified by conventional biochemical reaction. Out of 59 Mycobacterial isolates subjected to ICT for the detection of MPT 64 antigen, only 28 were identified as MTB. However, all the 30 isolates were correctly identified as MTB by PCR. Conclusion: Hence, PCR is essential for rapid differentiation of non-tuberculous Mycobacterium from MTB. False negative results seen with immunochromatographic MPT 64 antigen assay could be due to mutations within the mpt64 gene. Further studies are necessary to characterize these PCR-positive and immunochromatographic assay negative MTB isolates.

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

scholarly journals COVID-19, A New Outlook on Pandemic Symptoms and Diagnostic Approach

2022 ◽  
Vol 02 (01) ◽  
Author(s):  
María Fernanda Calderón Hernádez ◽  
Keyword(s):  
Diabetes Mellitus ◽  
Risk Factors ◽  
Polymerase Chain Reaction ◽  
Latin America ◽  
Diagnostic Methods ◽  
Chain Reaction ◽  
Specificity And Sensitivity ◽  
Polymerase Chain ◽  
Effective Diagnosis ◽  
Age And Sex

Background: The main objective of this research is to learn the symptoms that occur in this pathology, since we are currently still fighting COVID-19, because of this, it is important to keep us informed about the different diagnostic methods available, which help us reach an earlier and more effective diagnosis. Various articles have been compiled to identify as soon as possible the active cases and thus reduce the number of infections. Materials and methods: This research was conducted on the basis of scientific articles and books, related to COVID-19. Methods: This research was conducted based on 15 scientific articles and 3 books, related to COVID-19. Results: The most important risk factors are diabetes mellitus, hypertension, obesity, age and sex. The most common symptoms in Latin America are dry cough, fatigue, sore throat, and fever. The preferred diagnostic test for COVID-19 is the polymerase chain reaction for its specificity and sensitivity Conclusions: As a conclusion, the main objective of the research was achieved, which is to inform the reader about the most relevant symptoms of SARS-CoV-2 in order to improve the identification of suspected cases. Furthermore, we compare various diagnostic methods that exist to date and determine that PCR is the most specific and sensitive.

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