scholarly journals Protocol for bloodmeal identification in ticks using retrotransposon-targeted real time PCR

2021 ◽  
Author(s):  
Heidi Goethert
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Current Knowledge ◽  
Life Stage ◽  
Control Measures ◽  
Effective Control ◽  
Global Public Health ◽  
Public Health Burden ◽  
Intracellular Digestion ◽  
Bloodmeal Identification

Abstract Tick-borne infections are a global public health burden. Our ability to develop effective control measures relies on understanding the natural transmission cycles as well as the mode of exposure to humans. Our current knowledge of the relative importance of the hosts upon which tick vectors feed is based on indirect measurements, such as infestation indices. Bloodmeal identification can determine the host upon which a questing tick had fed in the previous life stage and is an unbiased method for incriminating reservoir hosts. Although bloodmeal identification has been utilized for mosquito ecology since the 1950s, the extended life-cycle of ticks and complete intracellular digestion of the bloodmeal make such analyses for ticks more complicated. We have recently developed a highly sensitive assay for identification of bloodmeals in ticks based upon real-time PCR amplification of mammalian retrotransposons. Although other PCR-based methods for bloodmeal analysis in ticks have been published previously, they have an inadequate success rate with field-collected ticks and have not been adopted for general use. We describe our methods in detail in order to make this assay widely accessible for use in other laboratories.

scholarly journals Molecular Survey of Vector-Borne Pathogens of Dogs and Cats in Two Regions of Saudi Arabia

Pathogens ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 25
Author(s):  
Abdullah D. Alanazi ◽  
Abdulaziz S. Alouffi ◽  
Mohamed S. Alyousif ◽  
Mohammad Y. Alshahrani ◽  
Hend H. A. M. Abdullah ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Real Time ◽  
Real Time Pcr ◽  
Bartonella Henselae ◽  
Public Awareness ◽  
Effective Control ◽  
Vector Borne Diseases ◽  
Babesia Vogeli ◽  
Vector Borne ◽  
First Time

Dogs and cats play an important role as reservoirs of vector-borne pathogens, yet reports of canine and feline vector-borne diseases in Saudi Arabia are scarce. Blood samples were collected from 188 free-roaming dogs and cats in Asir (70 dogs and 44 cats) and Riyadh (74 dogs), Saudi Arabia. The presence of Anaplasma spp., Bartonella spp., hemotropic Mycoplasma spp., Babesia spp., and Hepatozoon spp. was detected using a multiplex tandem real-time PCR. PCR-positive samples were further examined with specific conventional and real-time PCR followed by sequencing. Dogs from Riyadh tested negative for all pathogens, while 46 out of 70 dogs (65.7%) and 17 out of 44 cats (38.6%) from Asir were positive for at least one pathogen. Positive dogs were infected with Anaplasma platys (57.1%), Babesia vogeli (30%), Mycoplasma haemocanis (15.7%), and Bartonella henselae (1.4%), and cats were infected with Mycoplasma haemofelis (13.6%), Candidatus Mycoplasma haemominutum (13.6%), B. henselae (9.2%), and A. platys (2.27%), all of which are reported for the first time in Saudi Arabia. Co-infection with A. platys and B. vogeli was detected in 17 dogs (24.28%), while coinfections were not detected in cats. These results suggest that effective control and public awareness strategies for minimizing infection in animals are necessary.

Development and evaluation of real-time PCR assays for bloodmeal identification inCulicoidesmidges

2016 ◽  
Vol 30 (2) ◽  
pp. 155-165 ◽  
Author(s):  
M. R. VAN DER SAAG ◽  
X. GU ◽  
M. P. WARD ◽  
P. D. KIRKLAND
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Assays ◽  
Bloodmeal Identification

Transmission dynamics and control options for Echinococcus granulosus

Parasitology ◽  
2003 ◽  
Vol 127 (S1) ◽  
pp. S143-S158 ◽  
Author(s):  
P. R. TORGERSON ◽  
D. D. HEATH
Keyword(s):  
Echinococcus Granulosus ◽  
Disease Incidence ◽  
Effective Means ◽  
Public Health Problem ◽  
Control Measures ◽  
Transmission Dynamics ◽  
Effective Control ◽  
Global Public Health ◽  
Persistent Problem ◽  
Economic Output

Cystic echinococcosis, caused by the larval stage of Echinococcus granulosus, is a global public health problem. Whilst in a few localities, such as New Zealand, the parasite has been effectively controlled or even eradicated, in most endemic regions it remains a persistent problem. In some areas, such as the former Soviet Union, the disease incidence in humans has increased rapidly in recent years. It is important to have an understanding of the transmission dynamics, both between dogs and domestic livestock where the parasite maintains itself and from dogs to people. It is from this knowledge that effective control measures can be devised to reduce the prevalence of the parasite in animals and hence reduce the incidence of human disease. Mathematical models to describe the transmission of the parasite and the effects of different control strategies were first proposed over twenty years ago. Since then further information has been acquired, new technology has been developed and better computing technology has become available. In this review, we summarise these developments and put together a theoretical framework on the interpretation of surveillance information, how this affects transmission and how this information can be exploited to develop new intervention strategies for the control of the parasite. In particular, the parasite remains a persistent or re-emerging problem in countries of low economic output where resources for an intensive control programme, that has been successful in rich countries, are not available. By understanding of the transmission biology, including mathematical modelling, alternative and cost-effective means of control can be developed.

scholarly journals Evaluation of Real Time PCR for the diagnosis of Extrapulmonary Tuberculosis and comparison with AFB Microscopy among Bangladeshi population

2012 ◽  
Vol 2 (1) ◽  
pp. 26-32 ◽  
Author(s):  
MR Chowdhury ◽  
SMN Bari ◽  
KS Islam ◽  
AGM Rakibuzzaman ◽  
S Afrin
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Extrapulmonary Tuberculosis ◽  
Percutaneous Nephrostomy ◽  
The Body ◽  
Public Health Issue ◽  
Pcr Analysis ◽  
Global Public Health ◽  
Pcr Assay ◽  
Bangladeshi Population

Tuberculosis is a global public health issue and a disease burden in Africa and Asia. Bangladesh is one of the high burden countries in Southeast Asian region. Mycobacterium tuberculosis the causative agent of tuberculosis, is a devastating bacterium because it spreads person to person, acquired multiple antibiotic resistance and most importantly has the limitation in rapid diagnosis, specially in case of extrapulmonary infection. Here we showed Real-Time PCR assay with the primers targeting IS6110 is a better method than conventional AFB microscopy for the diagnosis of extrapulmonary tuberculosis. For the comparative study 99 extrapulmonary specimens from 9 different parts of the body were collected from suspected patients. Among those only 10 samples were positive, where 50% them were scanty positive in AFB microscopy, on the other hand 33 samples were positive in Real–Time PCR assay. All samples positive in AFB microscopy were also positive in Real-Time PCR assay with an additional 23 positive. Higher percentage of positive results were found in Real-Time PCR analysis in all the samples except Percutaneous nephrostomy (PCN) fluid and Pus. The sharp differences in the result indicate the effectiveness and of the Real-Time PCR assay over the conventional AFB microscopy. High precision and accuracy make it better choice as a diagnostic method for the diagnosis of extrapulmonary tuberculosis. DOI: http://dx.doi.org/10.3329/ijns.v2i1.10880 International Journal of Natural Sciences (2012), 2(1): 26-32

scholarly journals Development and Validation of a Real-Time PCR Assay for Rapid Detection of Candida auris from Surveillance Samples

2017 ◽  
Vol 56 (2) ◽  
Author(s):  
L. Leach ◽  
Y. Zhu ◽  
S. Chaturvedi
Keyword(s):  
Health Care ◽  
Real Time ◽  
Real Time Pcr ◽  
Multidrug Resistant ◽  
Effective Control ◽  
Pcr Assay ◽  
Candida Auris ◽  
Content Type ◽  
The Real ◽  
Positive Results

ABSTRACT Candida auris is an emerging multidrug-resistant yeast causing invasive health care-associated infection with high mortality worldwide. Rapid identification of C. auris is of primary importance for the implementation of public health measures to control the spread of infection. To achieve these goals, we developed and validated a TaqMan-based real-time PCR assay targeting the internal transcribed spacer 2 (ITS2) region of the ribosomal gene. The assay was highly specific, reproducible, and sensitive, with the detection limit of 1 C. auris CFU/PCR. The performance of the C. auris real-time PCR assay was evaluated by using 623 surveillance samples, including 365 patient swabs and 258 environmental sponges. Real-time PCR yielded positive results from 49 swab and 58 sponge samples, with 89% and 100% clinical sensitivity with regard to their respective culture-positive results. The real-time PCR also detected C. auris DNA from 1% and 12% of swab and sponge samples with culture-negative results, indicating the presence of dead or culture-impaired C. auris. The real-time PCR yielded results within 4 h of sample processing, compared to 4 to 14 days for culture, reducing turnaround time significantly. The new real-time PCR assay allows for accurate and rapid screening of C. auris and can increase effective control and prevention of this emerging multidrug-resistant fungal pathogen in health care facilities.

scholarly journals Development of a Taqman real-time PCR assay for rapid detection and quantification ofVibrio tapetisin extrapallial fluids of clams

PeerJ ◽  
2015 ◽  
Vol 3 ◽  
pp. e1484 ◽  
Author(s):  
Adeline Bidault ◽  
Gaëlle G. Richard ◽  
Cédric Le Bris ◽  
Christine Paillard
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Sea Water ◽  
Control Measures ◽  
Dilution Factor ◽  
Pcr Assay ◽  
Important Species ◽  
Quantitative Identification ◽  
Template Dna ◽  
Kinetics Of

The Gram-negative bacteriumVibrio tapetisis known as the causative agent of Brown Ring Disease (BRD) in the Manila clamVenerupis(=Ruditapes)philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification ofV. tapetisstrains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600TV. tapetisstrain. Quantification curves ofV. tapetisstrain seeded in filtered seawater (FSW) or extrapallial fluids (EF) samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detectV. tapetisstrains down to 1.125 101bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitorV. tapetisload both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections byV. tapetisand for designing appropriate control measures for aquaculture purposes.

scholarly journals Real-Time PCR Targeting Mosaic penA XXXIV for Prediction of Extended-Spectrum Cephalosporins Susceptibility in Clinical Neisseria Gonorrheae Isolates

2017 ◽  
Vol 4 (suppl_1) ◽  
pp. S70-S70
Author(s):  
Peera Hemarajata ◽  
Lisa Wong ◽  
Olusegun Soge ◽  
Romney Humphries ◽  
Jeffrey Klausner
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
False Negative ◽  
Rapid Test ◽  
Public Health Problem ◽  
Reference Laboratory ◽  
Global Public Health ◽  
The Us ◽  
Pcr Targeting

Abstract Background Antimicrobial-resistant Neisseria gonorrheae (NG) is a global public health problem, resulting in limited empirical treatment options. Due to increasing minimum inhibitory concentrations (MICs) of ESCs against NG in the US, it is critical that susceptibility to ESCs be monitored. Since few laboratories routinely perform culture and susceptibility testing for NG, there is a need for a rapid test to predict susceptibility to ESCs. More than 98% of isolates with decreased susceptibility to cefixime (CFM) in the US carry mosaic penA XXXIV. In this study, we developed a multiplex real-time PCR for mosaic penA XXXIV and previously validated gyrA to predict ESCs MICs and ciprofloxacin (CIP) susceptibility. Methods 150 NG isolates with known cefpodoxime (CPD), CFM, ceftriaxone (CRO) and CIP MICs were obtained from Neisseria Reference Laboratory at University of Washington and CDC Antimicrobial Resistance Bank. DNA extracted from culture was used in multiplex HybProbe real-time PCR on Lightcycler 480. gyrA was genotyped by melt curve and served as internal control, while presence of mosaic penA XXXIV was detected by selective amplification. Results All 32 (100%) CIP-susceptible and 118 (100%) CIP-resistant isolates, as determined by Clinical and Laboratory Standards Institute breakpoints, demonstrated wild-type and Ser91 mutant gyrA genotype, respectively. Melt curve genotyping demonstrated mosaic penA XXXIV melt patterns in 66/68 (97%) isolates with at least one ESC MIC above alert value set forth by the CDC (CPD and CFM MICs ≥0.25 µg/ml; CRO MIC ≥0.125), while all 82 (100%) isolates with ESC MICs under alert values did not amplify. The first of the 2 false-negative isolates had MICs above alert values for all ESCs tested and harbored IX mosaic type, while the second one had CRO MIC above alert value and harbored XII mosaic type. Both of these mosaic types did not share homology with mosaic penA XXXIV in the region targeted by the assay. Conclusion The mosaic penA XXXIV assay demonstrated 97% sensitivity and 100% specificity in predicting alert ESCs MIC values among clinical isolates tested, and was successfully multiplexed with gyrA assay. Clinical utility of this assay may be limited due to false negativity in isolates with non-XXXIV mosaic types, but it could serve as a useful surveillance tool for XXXIV mosaic. Disclosure R. Humphries, Roche: Consultant, Consulting fee

Rapid Quantitative Detection of Listeria monocytogenes in Salmon Products: Evaluation of Pre–Real-Time PCR Strategies

2005 ◽  
Vol 68 (7) ◽  
pp. 1467-1471 ◽  
Author(s):  
DAVID RODRÍGUEZ-LÁZARO ◽  
ANNA JOFRÉ ◽  
TERESA AYMERICH ◽  
MARGARITA GARRIGA ◽  
MARIA PLA
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Control Measures ◽  
Plate Count ◽  
Quantitative Detection ◽  
Promising Alternative ◽  
Fish Products ◽  
Smoked Fish ◽  
Seafood Processing

The spread and persistence of Listeria monocytogenes in smoked fish products and seafood processing factories are big concerns. Thus, the corresponding quality assurance programs must include adequate microbiological control measures. We evaluated eight different pre-PCR sample processing strategies to be coupled with a previously developed real-time PCR assay for the quantitative detection of L. monocytogenes in salmon products. The optimal pre-PCR procedure involved filtration and DNA purification with the use of a commercial kit. This strategy could detect 10 CFU of L. monocytogenes per g of smoked salmon and could quantify 1,000 CFU/g with excellent accuracy compared with the standard plate count method. Thus, this method could be a promising alternative for the quantitative detection of L. monocytogenes in smoked fish products and processing factories. This method could also detect the bacterium in raw salmon.

scholarly journals Real-Time PCR Detection of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum

2012 ◽  
Vol 78 (11) ◽  
pp. 3985-3991 ◽  
Author(s):  
Maria Teresa Martín ◽  
Rebeca Cobos ◽  
Laura Martín ◽  
Lorena López-Enríquez
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Control Measures ◽  
Economic Losses ◽  
Dna Purification ◽  
Time Saving ◽  
Content Type ◽  
Phaeomoniella Chlamydospora ◽  
Phaeoacremonium Aleophilum ◽  
Detection And Identification

ABSTRACTPhaeomoniella chlamydosporaandPhaeoacremonium aleophilumare the two main fungal causal agents of Petri disease and esca. Both diseases cause significant economic losses to viticulturalists. Since no curative control measures are known, proactive defensive measures must be taken. An important aspect of current research is the development of sensitive and time-saving protocols for the detection and identification of these pathogens. Real-time PCR based on the amplification of specific sequences is now being used for the identification and quantification of many infective agents. The present work reports real-time PCR protocols for identification ofP. chlamydosporaandP. aleophilum. Specificity was demonstrated against purified DNA from 60P. chlamydosporaisolates or 61P. aleophilumisolates, and no amplification was obtained with 54 nontarget DNAs. The limits of detection (i.e., DNA detectable in 95% of reactions) were around 100 fg forP. chlamydosporaand 50 fg forP. aleophilum. Detection was specific and sensitive forP. chlamydosporaandP. aleophilum. Spores ofP. chlamydosporaandP. aleophilumwere detected without the need for DNA purification. The established protocols detected these fungi in wood samples after DNA purification.P. chlamydosporawas detectable without DNA purification and isolation in 67% of reactions. The detection of these pathogens in wood samples has great potential for use in pathogen-free certification schemes.

scholarly journals What does soil-transmitted helminth elimination look like? Results from a targeted molecular detection survey in Japan

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Mitsuko Hasegawa ◽  
◽  
Nils Pilotte ◽  
Mihoko Kikuchi ◽  
Arianna R. Means ◽  
...  
Keyword(s):  
Real Time ◽  
Ascaris Lumbricoides ◽  
Real Time Pcr ◽  
Population Level ◽  
Molecular Techniques ◽  
Control Measures ◽  
Detection Methods ◽  
High Standards ◽  
Epidemiological Characteristics ◽  
Transmission Status

Abstract Background Japan is one of the few countries believed to have eliminated soil-transmitted helminths (STHs). In 1949, the national prevalence of Ascaris lumbricoides was 62.9%, which decreased to 0.6% in 1973 due to improvements in infrastructure, socioeconomic status, and the implementation of national STH control measures. The Parasitosis Prevention Law ended in 1994 and population-level screening ceased in Japan; therefore, current transmission status of STH in Japan is not well characterized. Sporadic cases of STH infections continue to be reported, raising the possibility of a larger-scale recrudescence of STH infections. Given that traditional microscopic detection methods are not sensitive to low-intensity STH infections, we conducted targeted prevalence surveys using sensitive PCR-based assays to evaluate the current STH-transmission status and to describe epidemiological characteristics of areas of Japan believed to have achieved historical elimination of STHs. Methods Stool samples were collected from 682 preschool- and school-aged children from six localities of Japan with previously high prevalence of STH. Caregivers of participants completed a questionnaire to ascertain access to water, sanitation and hygiene (WASH), and potential exposures to environmental contamination. For fecal testing, multi-parallel real-time PCR assays were used to detect infections of Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale and Trichuris trichiura. Results Among the 682 children, no positive samples were identified, and participants reported high standards of WASH. Conclusions To our knowledge, this is the first STH-surveillance study in Japan to use sensitive molecular techniques for STH detection. The results suggest that recrudescence of STH infections has not occurred, and that declines in prevalence have been sustained in the sampled areas. These findings suggest that reductions in prevalence below the elimination thresholds, suggestive of transmission interruption, are possible. Additionally, this study provides circumstantial evidence that multi-parallel real-time PCR methods are applicable for evaluating elimination status in areas where STH prevalence is extremely low.

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