Diagnosis and epidemiological studies of human metapneumovirus using real-time PCR

Background Elimination and control of Schistosoma japonicum, the most virulent of the schistosomiasis-causing blood flukes, requires the development of sensitive and specific diagnostic tools capable of providing an accurate measurement of the infection prevalence in endemic areas. Typically, detection of S. japonicum has occurred using the Kato-Katz technique, but this methodology, which requires skilled microscopists, has been shown to radically underestimate levels of infection. With the ever-improving capabilities of next-generation sequencing and bioinformatic analysis tools, identification of satellite sequences and other highly repetitive genomic elements for use as real-time PCR diagnostic targets is becoming increasingly common. Assays developed using these targets have the ability to improve the sensitivity and specificity of results for epidemiological studies that can in turn be used to inform mass drug administration and programmatic decision making. Methodology/Principal findings Utilizing Tandem Repeat Analyzer (TAREAN) and RepeatExplorer2, a cluster-based analysis of the S. japonicum genome was performed and a tandemly arranged genomic repeat, which we named SjTR1 (Schistosoma japonicum Tandem Repeat 1), was selected as the target for a real-time PCR diagnostic assay. Based on these analyses, a primer/probe set was designed and the assay was optimized. The resulting real-time PCR test was shown to reliably detect as little as 200 ag of S. japonicum genomic DNA and as little as 1 egg per gram of human stool. Based on these results, the index assay reported in this manuscript is more sensitive than previously published real-time PCR assays for the detection of S. japonicum. Conclusions/Significance The extremely sensitive and specific diagnostic assay described in this manuscript will facilitate the accurate detection of S. japonicum, particularly in regions with low levels of endemicity. This assay will be useful in providing data to inform programmatic decision makers, aiding disease control and elimination efforts.

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Detection of Leishmania infantum in Lutzomyia longipalpis captured in Campo Grande, MS

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612014049 ◽

2014 ◽

Vol 23 (2) ◽

pp. 269-273 ◽

Cited By ~ 11

Author(s):

Rodrigo Casquero Cunha ◽

Renato Andreotti ◽

Marlon Cezar Cominetti ◽

Elaine Araújo Silva

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Urban Areas ◽

Leishmania Infantum ◽

Epidemiological Studies ◽

Lutzomyia Longipalpis ◽

Forest Reserves ◽

Primary Vector ◽

The City ◽

Genus Leishmania

Leishmaniasis is a zoonotic disease caused by protozoa of the genus Leishmania (Ross, 1903) and is the focus of considerable attention in human and veterinary medicine. In the city of Campo Grande, MS, the causative agent of visceral leishmaniasis is Leishmania infantum (= L. chagasi) primary vector, comprising approximately 92.9% of the local sandfly population, is Lutzomyia longipalpis. The aim of this work was to compare real-time PCR with PCR as a tool for the detection of the kinetoplast DNA (kDNA) of L. infantum in sandflies. Sandflies of this species were caught, and a total of 38 samples with 1-4 individuals in each sample were obtained; these were distributed across 13 districts and divided between seven urban areas of the city of Campo Grande, MS. Three positive samples were found by PCR and, when using real-time PCR, this was able to detect the presence of this agent in 6 of the 13 districts sampled, which were all located on the outskirts of the city, where indicates the greater enzootic potential of these regions, as they are closer to natural forest reserves. We conclude that real-time PCR can be used for epidemiological studies of L. infantum.

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Trimodal GSTT1 and GSTM1 Genotyping Assay by Real-Time PCR

The International Journal of Biological Markers ◽

10.1177/172460080502000201 ◽

2005 ◽

Vol 20 (2) ◽

pp. 81-86 ◽

Cited By ~ 8

Author(s):

I. Girault ◽

R. Lidereau ◽

I. Bièche

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Null Allele ◽

Epidemiological Studies ◽

Published Data ◽

Real Time Quantitative Pcr ◽

Albumin Gene ◽

The Mean ◽

Pcr Assays ◽

Risk Of Cancer

The GSTT1 and GSTM1 genes are characterized by the existence of a GST*0 null allele responsible for a lack of enzyme activity, with the respective null genotypes GSTT1*0/0 and GSTM1*0/0. The three resulting genotypes (GSTs*1/1, *1/0 and *0/0) are associated with a trimodal distribution of glutathione-conjugator activity. Previous epidemiological studies have only evaluated the cancer risk associated with the GST null genotype relative to the two GST carrier geno-® types (GSTs1*1/1 and *1/0). We developed GSTT1 and GSTM1 TaqMan real-time quantitative PCR assays to discriminate each of the three genotypes, with the albumin gene (ALB) as reference. The mean NGSTT1*1/1 value was 1.0 (95% confidence interval 0.80–1.20). The mean NGSTT1*1/0 value was 0.48 (95% CI 0.36–0.60). One (3.4%) of the 29 DNA samples yielded the GSTM1*1/1 genotype (NGSTM1*1/1 = 1), a frequency in keeping with the Hardy-Weinberg distribution. The mean NGSTM1*1/0 value was 0.50 (95% CI 0.42–0.58). All GSTT1*0/0 and GSTM1*0/0 samples yielded NGST values of 0 (Ct = 40); the frequencies of these genotypes (27.6% and 55.2%, respectively) were in keeping with published data. The GSTT1 and GSTM1 real-time PCR assays described here unambiguously discriminate each of the three existing genotypes which should be valuable for assessing the relative risk of cancer associated with each of the three GST genotypes.

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Detection and Monitoring of Greeneria uvicola and Colletotrichum acutatum Development on Grapevines by Real-Time PCR

Plant Disease ◽

10.1094/pdis-07-10-0537 ◽

2011 ◽

Vol 95 (3) ◽

pp. 298-303 ◽

Cited By ~ 13

Author(s):

Suren K. Samuelian ◽

Lindsay A. Greer ◽

Sandra Savocchia ◽

Christopher C. Steel

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Epidemiological Studies ◽

Colletotrichum Acutatum ◽

Internal Transcribed Spacer Region ◽

Bitter Rot ◽

Pcr Method ◽

Polymerase Chain ◽

Species Specific ◽

And Control

Bitter rot (Greeneria uvicola) and ripe rot (Colletotrichum acutatum, syn. C. simmondsii) occur frequently in subtropical grape-growing regions of Australia, where they cause yield loss and bitter taints in wine. To further advance the epidemiological studies of G. uvicola and C. acutatum and contribute toward their effective management and control, a rapid and reliable species-specific real-time polymerase chain reaction (PCR) method was developed based on the polymorphic portion of the internal transcribed spacer region of the two fungi. It was found that, within 6 to 8 h postinoculation, the assay could detect as little as 20 fg of genomic DNA and 10 conidia for both species. Artificially and naturally infected grape inflorescences and mature berries were analyzed by both conventional plating methods and real-time PCR. Fungal presence was demonstrated on all plant material but development was observed only on mature berries. The results demonstrate that the real-time PCR technique is a highly specific, rapid, and sensitive method that can be used to detect and study the dynamics of G. uvicola and C. acutatum during different stages of infection and on different grape tissues.

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The Molecular Epidemiology of Cystic and Alveolar Echinococcosis in Southeast Turkey

Iranian Journal of Parasitology ◽

10.18502/ijpa.v16i2.6284 ◽

2021 ◽

Author(s):

Fadime Eroglu ◽

Mehmet Dokur ◽

Yüksel Ulu

Keyword(s):

Infectious Diseases ◽

Molecular Epidemiology ◽

Real Time ◽

Real Time Pcr ◽

Environmental Changes ◽

Phylogenetic Analyses ◽

Geographical Area ◽

Epidemiological Studies ◽

Genotype 3 ◽

Southeast Region

Background: The migration of humans and climatic and environmental changes cause the emergence of infectious diseases. This study aimed to investigate the changes in the molecular epidemiology of the echinococcosis in the southeast region of Turkey after migrations. Methods: Overall, 159 tissues samples were taken from suspected cases of echinococcosis at the Kilis State Hospital in the southeast region of Turkey. All of the tissues samples were analyzed using histopathology methods, PCR, Real-time PCR methods, DNA sequencing, and phylogenetic analyses in laboratories. Results: The positivity values of the histopathology, the polymerase chain reaction, and the Real-time PCR methods were found to be 14.5% (23/159), 15.7% (25/159), and 16.9% (27/159), respectively. 32.0 % (8/25) E. multilocularis of Echinococcus isolates and 68% (17/25) E. granulosus of Echinococcus isolates were identified using PCR methods. 58.8% (10/17) of the E. granulosus isolates were found to be Genotype 1% and 41.2% (7/17) E. granulosus isolates were found to be Genotype 3. Conclusion: Molecular methods play an important role in the epidemiology, treatment, and diagnosis of diseases. Increasing immigration in a geographical area may create social, economic, and health problems in that area. For this reason, epidemiological studies of infectious diseases should be updated in areas with immigration.

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