Toxocara DNA amplification in serum and tissue samples in BALB/c mice

AimsThe identification of actionable DNA mutations associated with a patient’s tumour is critical for devising a targeted, personalised cancer treatment strategy. However, these molecular analyses are typically performed using tissue obtained via biopsy, which involves substantial risk and is often not feasible. In addition, biopsied tissue does not always reflect tumour heterogeneity, and sequential biopsies to track disease progression (eg, emergence of drug resistance mutations) are not well tolerated. To overcome these and other biopsy-associated limitations, we have developed non-invasive ‘liquid biopsy’ technologies to enable the molecular characterisation of a patient’s cancer using peripheral blood samples.MethodsThe Target Selector ctDNA platform uses a real-time PCR-based approach, coupled with DNA sequencing, to identify cancer-associated genetic mutations within circulating tumour DNA. This is accomplished via a patented blocking approach suppressing wild-type DNA amplification, while allowing specific amplification of mutant alleles.ResultsTo promote the clinical uptake of liquid biopsy technologies, it is first critical to demonstrate concordance between results obtained via liquid and traditional biopsy procedures. Here, we focused on three genes frequently mutated in cancer: EGFR (Del19, L858, and T790), BRAF (V600) and KRAS (G12/G13). For each Target Selector assay, we demonstrated extremely high accuracy, sensitivity and specificity compared with results obtained from tissue biopsies. Overall, we found between 93% and 96% concordance to blinded tissue samples across 127 clinical assays.ConclusionsThe switch-blocker technology reported here offers a highly effective method for non-invasively determining the molecular signatures of patients with cancer.

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Detection ofBorrelia burgdorferi by DNA amplification in synovial tissue samples from patients with lyme arthritis

Arthritis & Rheumatism ◽

10.1002/art.1780390505 ◽

1996 ◽

Vol 39 (5) ◽

pp. 736-745 ◽

Cited By ~ 76

Author(s):

Beno??t Jaulhac ◽

Isabelle Chary-Valckenaere ◽

Jean Sibilia ◽

Rose-Marie Javier ◽

Yves Piémont ◽

...

Keyword(s):

Synovial Tissue ◽

Dna Amplification ◽

Lyme Arthritis ◽

Tissue Samples

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Prevalence of Toxoplasma gondii in retail fresh meat products from free-range chickens in Spain

Journal of Veterinary Research ◽

10.2478/jvetres-2021-0052 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

María Jesús Gracia Salinas ◽

Cristina Escolano Campos ◽

María Paz Peris Peris ◽

Nabil Halaihel Kassab

Keyword(s):

Toxoplasma Gondii ◽

Meat Products ◽

Oral Intake ◽

Dna Amplification ◽

Specific Sequence ◽

Tissue Samples ◽

High Exposure ◽

Free Range ◽

Warm Blooded Animal ◽

Postnatal Infection

Abstract Introduction Toxoplasma gondii is one of the most prevalent zoonotic protozoan parasites worldwide and affects the vast majority of warm-blooded animal species, including humans. Postnatal infection in humans occurs through the ingestion of sporulated T. gondii oocysts or via the oral intake of parasite tissue cysts during the consumption of raw or undercooked meat. In this regard, given their high exposure to oocysts, chickens (Gallus domesticus) raised on the ground constitute a potential source of T. gondii. Material and Methods For the first time in Spain, a survey was undertaken in commercial retail free-range poultry. A total of 50 thighs from different animals were analysed. The samples were homogenised and an acid pepsin digestion procedure was applied prior to molecular analysis. Toxoplasma gondii DNA was isolated from meat by qPCR. Two sets of primers were used for DNA amplification targeting the specific sequence of a 529 bp repeat element and another set of primers was utilised for the surface antigen protein-1 gene. Results DNA extracted from 5 out of 50 tissue samples was positive for both genes by qPCR amplification. Conclusion The 10% prevalence of Toxoplasma infection found in commercial free-range chickens raises public health issues.

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A preliminary survey for avian pathogens in Columbiform birds on Socorro Island, Mexico.

Pacific Conservation Biology ◽

10.1071/pc110011 ◽

2011 ◽

Vol 17 (1) ◽

pp. 11 ◽

Cited By ~ 5

Author(s):

Saturnino Yanga ◽

Juan E Martinez-Gomez ◽

Ravinder N M Sehgal ◽

Patricia Escalante ◽

Frank Carlos Camacho ◽

...

Keyword(s):

Dna Amplification ◽

Cloacal Swab ◽

Mourning Dove ◽

Tissue Samples ◽

Zenaida Macroura ◽

Trichomonas Gallinae ◽

Socorro Island ◽

Disease Risks ◽

Mourning Doves ◽

Avian Pox

To assess the potential disease risks posed by resident Columbiformes to the reintroduction of the Socorro Dove Zenaida graysoni to Socorro Island, Mexico, the endemic Socorro Ground Dove Columbina passerina socorrensis and the recently arrived Mourning Dove Zenaida macroura, were screened for ecto- and endoparsites, haemosporidia, Trichomonas gallinae, Chlamydophila psittaci and avian pox. All of the Mourning Doves and Socorro Ground Doves sampled appeared healthy upon capture. We detected Haemoproteus spp. in 88% of Mourning Dove and 30% of Socorro Ground Dove samples using microscopy. Two polymerase chain reaction (PCR) DNA amplification methods detected either Haemoproteus spp. or Plasmodium spp. Pooling results from both tests yielded positives in 100% of the Mourning Doves and 52% of the Socorro Ground Doves. A nested PCR detected Leucocytozoon spp. in 94% of the Mourning Doves and 61% of the Socorro Ground Doves sampled. Thus, at least two genera of haemosporidia are present in columbids of Socorro Island. Microscopy for T. gallinae yielded positives in 33% of Mourning Dove and 30% of Socorro Ground Dove samples. C. psittaci was not detected using PCR on either cloacal swab samples or tissue samples from tested Mourning Doves or Socorro Ground Doves. Necropsies revealed neither lesions indicative of the wet form of avian pox, nor internal lesions associated with trichomoniasis. These results suggest that Socorro Doves selected for reintroduction should be screened carefully to evaluate potential immunological challenges by native haemosporidians and to avoid introduction of other diseases apparently absent from native Columbiformes on Socorro Island.

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Multiplexed detection of RNA using MERFISH and branched DNA amplification

10.1101/505784 ◽

2018 ◽

Author(s):

Chenglong Xia ◽

Hazen P Babcock ◽

Jeffrey R Moffitt ◽

Xiaowei Zhuang

Keyword(s):

Detection Efficiency ◽

Dna Amplification ◽

Spot Size ◽

Tissue Samples ◽

Rna Molecules ◽

Simultaneous Imaging ◽

Spatially Resolved ◽

Branched Dna ◽

Cellular Environment ◽

Fish Samples

Multiplexed error-robust fluorescence in situ hybridization (MERFISH) allows simultaneous imaging of numerous RNA species in their native cellular environment and hence spatially resolved single-cell transcriptomic measurements. However, the relatively modest brightness of signals from single RNA molecules can become limiting in a number of important applications, such as increasing the imaging throughput, imaging shorter RNAs, and imaging samples with high degrees of background, such as some tissue samples. Here, we introduce a branched DNA (bDNA) amplification approach for MERFISH measurements. This approach produces a drastic signal increase in RNA FISH samples without increasing the fluorescent spot size for individual RNAs or increasing the variation in brightness from spot to spot. Using this approach in combination with MERFISH, we demonstrated RNA imaging and profiling with a near 100% detection efficiency. We further demonstrated that signal amplification improves MERFISH performance when fewer FISH probes are used for each RNA species, which should allow shorter RNAs to be imaged. We anticipate that the combination of bDNA amplification with MERFISH should facilitate many other applications and extend the range of biological questions that can be addressed by this technique in both cell culture and tissues.

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Exploratory study of histopathological characteristics of cervical cancer in an African (Tanzania) population.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e16530 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e16530-e16530

Author(s):

Sara Bravaccini ◽

Elisa Chiadini ◽

Maurizio Puccetti ◽

Patrizia Serra ◽

Nestory Andrew Masalu ◽

...

Keyword(s):

Cervical Cancer ◽

Invasive Cervical Cancer ◽

Dna Amplification ◽

Hpv Infection ◽

African Women ◽

Cancer Tissue ◽

Hpv 16 ◽

Tissue Samples ◽

Hpv Test

e16530 Background: In Tanzania, cervical cancer is the leading cause of death after breast cancer, with the majority of tumors diagnosed at a very advanced stage. Little information is available on the characteristics of cervical cancer in Sub-Saharan African women. We aimed to evaluate histopathological features of cervical cancers and relative margins in a group of African women and to characterize HPV infection. Methods: Tissue samples from32 Tanzanian women with invasive cervical cancer were evaluated in the Biosciences Laboratory of our Institute (IRCCS IRST, Meldola, Italy) as part of a global cancer control project currently ongoing in close cooperation with the Bugando Medical Center of Mwanza, Tanzania. Results: 24/32 (75%) women had squamous carcinoma and 4/32 (12.5%) had adenocarcinoma of the cervix. The HPV test was performed in the entire series but DNA amplification by pyrosequencing was only possible in 27/32 (84.4%). The presence of HPV infection was confirmed in 26/27 (96.3%) cases in both tumor and relative margins. Typization of the virus revealed that a high percentage of the women had HPV 16 virus of whom 12 (46.2 %) had African type 1 and 4 (15.4%) African type 2. Conclusions: A major problem in the cancer tissue samples from the Tanzanian patients was the poor evaluability of DNA amplification due to suboptimal fixation which compromised tissue morphology. Our preliminary study shows that cervical cancer in Tanzanian women is characterized by a very high frequency of HPV-positive tumors, with a high prevalence of HPV 16 African type 1 and 2 infections. The substantial number of HPV infections could explain the high rate of cervical cancer and subsequent mortality in this African population. [Table: see text]

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Molecular tools for phytoplankton monitoring samples

10.1101/339655 ◽

2018 ◽

Author(s):

Bárbara Frazão ◽

Alexandra Silva

Keyword(s):

Dna Extraction ◽

Dna Polymerases ◽

Life Sciences ◽

Dna Amplification ◽

Cell Number ◽

Molecular Tools ◽

Direct Pcr ◽

Tissue Samples ◽

Thermofisher Scientific ◽

Phytoplankton Monitoring

AbstractHABs can have severe impacts in fisheries or human health by the consumption of contaminated bivalves. Monitoring assessment (quantitative and qualitative identification) of these organisms, is routinely accomplished by microscopic identification and counting of these organisms. Nonetheless, molecular biology techniques are gaining relevance, once these approaches can easily identify phytoplankton organisms at species level and even cell number quantifications. This work tests 12 methods/kits for genomic DNA extraction and seven DNA polymerases to determine which is the best method for routinely use in a common molecular laboratory, for phytoplankton monitoring samples analyses. From our work, Direct PCR master mix for tissue samples, proved to be the most adequate by its velocity of processivity, practicability, reproducibility, sensitiveness and robustness. However, brands such as Omega Biotek, GRISP, Qiagen and MP Biomedicals also showed good results for conventional DNA extraction as well as all the Taq brands tested (GRISP, GE Healthcare Life Sciences, ThermoFisher Scientific and Promega). Lugol’s solution, with our tested kits did not show negative interference in DNA amplification. The same can be said about mechanical digestion, with no significant differences among kits with or without this homogenization step.

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First Isolation and Molecular Characterization of Pseudorabies Virus Detected in Turkey

10.21203/rs.3.rs-659500/v1 ◽

2021 ◽

Author(s):

Gizem Aytoğu ◽

Eda B. Toker ◽

Ozkan Yavas ◽

Berfin Kadiroglu ◽

Ozer Ates ◽

...

Keyword(s):

Molecular Characterization ◽

Pseudorabies Virus ◽

Clinical Signs ◽

Dna Amplification ◽

Vero Cells ◽

Virus Genome ◽

Buffy Coat ◽

Fresh Tissue ◽

Tissue Samples

Abstract Pigs are the main host species for the pseudorabies virus, while it causes fatal encephalitis in many species including human being. The objective of this article is to report the first clinical case of pseudorabies as well as isolation and molecular characterization of the virus from a hunting dog in Bursa province, Turkey. The dog showing clinical signs including pruritus and neurological signs as stumbling and inability to stand up compatible with pseudorabies. The virus isolate was obtained from supernatant of fresh tissue samples from cerebellum, cornu ammonis, spleen, salivary gland, and conjunctival swab, as well as serum, and buffy coat samples. The glycoprotein C region is targeted for viral DNA amplification. Pseudorabies virus genome was detected both in fresh tissues and supernatants of 3rd passage on Vero cells. Number of PCR positive samples were dramatically increased after having cell culture inoculations. Genome sequencing revealed that the strain (Bursa-10303) isolated in Turkey where a non-endemic area is for the virus is dropped into clade A. This study confirms the presence of psedorabies infection in the wild life reservoirs in Turkey. Future studies may clarify the importance of the infection for Turkey where there is no common pig production.

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Minimally invasive blood sampling method for genetic studies on Gopherus tortoises

Animal Biodiversity and Conservation ◽

10.32800/abc.2015.38.0031 ◽

2015 ◽

Vol 38 (1) ◽

pp. 31-35 ◽

Cited By ~ 1

Author(s):

L. M. García–Feria ◽

◽

C. A. Ureña–Aranda ◽

A. Espinosa de los Monteros ◽

◽

...

Keyword(s):

Minimally Invasive ◽

Dna Amplification ◽

Molecular Study ◽

Invasive Technique ◽

Minimal Amount ◽

Medical Monitoring ◽

Tissue Samples ◽

Wild Fauna ◽

Cell Sources ◽

Handling Methods

Obtaining good quality tissue samples is the first hurdle in any molecular study. This is especially true for studies involving management and conservation of wild fauna. In the case of tortoises, the most common sources of DNA are blood samples. However, only a minimal amount of blood is required for PCR assays. Samples are obtained mainly from the brachial and jugular vein after restraining the animal chemically; or from conscious individuals by severe handling methods and clamping. Herein, we present a minimally invasive technique that has proven effective for extracting small quantities of blood, suitable for genetic analyses. Furthermore, the samples obtained yielded better DNA amplification than other cell sources, such as cloacal epithelium cells. After two years of use on wild tortoises, this technique has shown to be harmless. We suggest that sampling a small amount of blood could also be useful for other types of analyses, such as physiologic and medical monitoring.

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Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

Pesquisa Veterinária Brasileira ◽

10.1590/s0100-736x2012000800006 ◽

2012 ◽

Vol 32 (8) ◽

pp. 715-720 ◽

Cited By ~ 2

Author(s):

Paula R. Almeida ◽

Caroline P. Andrade ◽

Laura L. Almeida ◽

Luiz G.S. Oliveira ◽

Luiza A. Castro ◽

...

Keyword(s):

Dna Amplification ◽

Mycoplasma Hyopneumoniae ◽

Ffpe Tissue ◽

Tissue Samples ◽

Histological Features ◽

Frozen Tissue ◽

Ffpe Samples ◽

Formalin Fixed Paraffin Embedded ◽

Ffpe Tissues ◽

Formalin Fixed

The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC) and polymerase chain reaction (PCR) or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-fixed and paraffin-embedded (FFPE) tissue. However, the formalin fixation process often inhibits DNA amplification. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs) or frozen (tissue pieces). M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efficiency of PCR could be compromised under these conditions.

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