Name: the lambda select cii mutation detection system
Uploaded: 2018-04-26T15:00:00
Description: Watch this Scientific Journal Video about The Lambda Select cII Mutation Detection System at JoVE.com

Мы хотели бы признать вклад всех коллег и партнеров для наших оригинальных исследований, результаты которого были переданы в этой рукописи (для примера). Работа авторов поддерживается за счет субсидий из Национального Института стоматологии и челюстно-лицевой исследования национальных институтов здравоохранения (1R01DE026043) для AB и из университета California Tobacco-Related программы исследований болезни (AB TRDRP-26IR-0015) и ST (TRDRP-25IP-0001). Авторы данного исследования было никакой роли в дизайн исследования, сбор данных, анализ данных, интерпретация данных, написание доклада, или решение представить для публикации.

1. геномной ДНК изоляции от мыши эмбриональных фибробластов
Примечание: Основная мышь эмбриональных фибробластов изолированы от эмбрионов, полученных от BB трансгенных мышей C57BL/6 генетическим фоном, согласно опубликованной протокол53. Исходным материалом для...

Выберите cII пробирного λ используется для обнаружения мутаций в ИСИ трансген, оправился от геномной ДНК клеток, полученных от органов/тканей BB грызунов3. Геном этих трансгенных животных содержит несколько копий тандем хромосомно комплексной λLIZ Трансфер вектора, ...

Широкий спектр трансгенных животных моделей и систем обнаружения мутации были разработаны для мутагенность тестирования канцерогенов в mammalian клетках. Из них трансгенных Big Blue (далее именуемое BB) мышах и λ выберите cII мутации системы обнаружения использовались для экспериментов мутагенность этой группой и многих других исследовательских групп во всем мире1,2, 3,4,5,6,,78,9. За последние 16 лет, мы исследовали мутагенные эффекты различных химических или физических агентов, используя эти трансгенных животных или их соответствующих культур клеток эмбриональных фибробластов, относились с тест соединения и впоследствии проанализированы фенотип и генотип cII трансген λ выберите cII пробирного и секвенирования ДНК, соответственно10,11,12,13,14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24. геном этих трансгенных животных содержит бактериофага вектор Трансфер λ (λLIZ) интегрированы на хромосоме 4 как мульти копирования голова хвост concatemer1,2,25. ΛLIZ Трансфер вектор осуществляет два мутационного репортер генов, а именно ЗСБН и cII трансгенов1,2,25,26,27, 28,29,30,,3132,33,34,,3536, 37,,3839,40,41,42,,4344,45 , 46 , 47. пробирного выберите cII λ основывается на восстановление λLIZ Трансфер векторов геномной ДНК клеток, полученных от органов/тканей трансгенных животных1,2,25 . Восстановленный λLIZ Трансфер векторов затем упаковываются в λ Фаговые руководители способны заражать узла индикатор Escherichia coli. Впоследствии зараженных бактерий выращивают под селективного условий для оценки и анализа мутаций в ИСИ трансген1,3.
Здесь мы описываем подробный протокол для assay выберите cII λ, который состоит из изоляции genomic дна от клеток/органов трансгенных животных рассматривается в vitro/в vivo с тест соединения, Поиск λLIZ Трансфер векторов от genomic дна упаковка векторов в инфекционных λ бактериофаги, инфекции принимающей E. coli с бактериофагов, выявление cII-мутанты селективного условиях для определения частоты мутантных cII , и Анализ последовательности ДНК установить спектра мутаций cII . Протокол могут быть применены к трансгенные мыши/крыса клеток культур рассматривается в пробирке с агентом химические/физические интерес, или тканей/органы соответствующих животных обрабатывают в vivo с испытаний химических/агент1, 2,4,48,49,50,,5152. Схематическое представление выберите cII пробирного λ показан на рисунке 1.

<table>
	<tbody>
		<tr>
			<td>Agar</td>
			<td>MO Bio Laboratories, Inc.</td>
			<td>12112-05</td>
			<td>Bacteriological grade</td>
		</tr>
		<tr>
			<td>BigDye Terminator v3.1 Cycle Sequencing Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>4337455</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Casein Peptone</td>
			<td>Alfa Aesar</td>
			<td>H26557</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Gelatine</td>
			<td>J. T. Baker</td>
			<td>2124-01</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP 229-1 / M-13750</td>
			<td>None</td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Fisher Scientific</td>
			<td>BP 9724-500</td>
			<td>None</td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>8104</td>
			<td>50 PCR purification reactions</td>
		</tr>
		<tr>
			<td>Sodium Acetate Trihydrate</td>
			<td>Fisher Scientific</td>
			<td>M-15756</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Taq5000 DNA Polymerase&#160;</td>
			<td>Qiagen</td>
			<td>201207</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Thiamine Hydrochloride</td>
			<td>Macron Fine Chemicals</td>
			<td>2722-57</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Transpack Packaging Extract</td>
			<td>Stratagene Corp., Acquired by BioReliance | Sigma-Aldrich Corp.</td>
			<td>200223</td>
			<td>50 packaging reactions</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP 152-1 / EC 201-064-4</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Trypton</td>
			<td>Biosciences</td>
			<td>RC-110</td>
			<td>None</td>
		</tr>
	</tbody>
</table>

the lambda select cii mutation detection system

Ряд трансгенных животных моделей и систем обнаружения мутации были разработаны для мутагенность тестирования канцерогенов в mammalian клетках. Из них трансгенных мышей и лямбда (λ) выберите cII мутации системы обнаружения были заняты на мутагенность экспериментов на многих исследовательских групп во всем мире. Здесь мы описываем подробный протокол для лямбда выбрать assay cII мутации, которые могут быть применены для культивируемых клеток трансгенных мышей/крыс или соответствующих животных обрабатывают с агентом химические/физические интерес. Протокол состоит из следующих этапов: (1) изоляции геномной ДНК из клеток или органов/тканей трансгенных животных рассматривается в vitro или в естественных условиях, соответственно, с тест соединения; (2) восстановление вектора Трансфер лямбда, перевозящих мутационного Репортер ген (то есть, cII трансген) от геномной ДНК; (3) упаковки спасли векторов в инфекционных бактериофагов; (4) заражающий принимающей бактерии и культивирования условиях выборочный разрешить распространение мутаций индуцированных ИСИ ; и (5) забил cII-мутанты и ДНК последовательности анализа для определения частоты мутантных cII и спектра мутаций, соответственно.

В зависимости от распределения данных параметрические и непараметрические тесты используются для определения значимости различий в частоте мутант cII между группами лечения и управления (т.е., индуцированной против спонтанной частоты мутантных) . Производит...

Watch this Scientific Journal Video about The Lambda Select cII Mutation Detection System at JoVE.com

The Lambda Select cII Mutation Detection System

Лямбда выбрать cII мутации системы обнаружения

Мы описываем подробный протокол для assay лямбда выбрать мутации cII в культивируемых клеток трансгенных грызунов или соответствующих животных, лечение с агентом химические/физические интерес. Такой подход широко использовался для тестирования мутагенность канцерогенов в mammalian клетках.

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of ...

The overall goal of this protocol is to visualize step-wise procedures involved in the Lambda Select cII mutation assay in cultured cells of transgenic rodents or the corresponding animals treated with chemical and physical agents of interest. This test method can help answer key questions in the field of mutation research such as, is a test compound mutagenic? And if so, how potent of a mutagen is it?Does it induce sequence-specific mutations? The main advantage of this technique is that it is a minimal to virtually any test compound. The method can be used for mutagenicity testing in mammalian cells using a chromosomally integrated reporter gene.To constitute a single packaging reaction, add five micrograms of genomic DNA in a micro-centrifuge tube containing the first reaction mix. Then incubate the tube at 30 degrees Celsius for 90 minutes. Post-incubation, add 12 micro-liters of the second reaction mix to the tube and leave for another 90 minutes at 30 degrees Celsius.After 90 minutes, add 1.1 milliliters of SM buffer to the tube. Then vigorously vortex the tube with the packaged DNA at room temperature for about 10 seconds. Quickly give a pulse spin in the micro-centrifuge.Then leave the tube on ice until use. Next, add 50 microliters of chloroform for each milliliter of packaged DNA. If the sample will not be processed the same day, then vortex the sample gently and store at four degrees Celsius up to two weeks.Prepare 16 sterile round bottom tubes and 16 TB1 agar plates for a single packaged DNA sample. Use ten tubes for screening and six for titering. Then aliquot 300 microliters of G1250 plating culture in each round bottom tube.Next, to titer, add 10 microliters of packaged DNA to 990 microliters of SM buffer and 1:100 dilution. Then vortex the sample. Add 20 or 100 microliters of the 1:100 DNA solution to each of the three titer 20 or titer 100 tubes respectively.For screening purpose, add 100 microliters of the undiluted packaged DNA sample to each of the 10 screening tubes. Process all of the 16 titering and screening tubes. Vortex the tubes for 10 seconds to mix the components.Then incubate the tubes at room temperature for 30 minutes for the host E.coli to absorb the phages. Next, add 2.5 milliliters of molten TB1 top agar. Maintain that 55 degree Celsius to the appropriate titering and screening tubes.After adding, immediately vortex the tubes gently. Now, pour the TB1 agar to the appropriate titer or screening TB1 agar plate. Allow the plates to stand for 15 to 30 minutes at room temperature.Once the agar has solidified, invert and leave all the 10 screening plates in the stationary incubator at 24 degrees Celsius for 46 to 48 hours. Leave the remaining six titer plates in a stationary incubator at 37 degrees Celsius for overnight or a day. Following the incubation at 37 degrees Celsius, count the number of plaques formed in all the 3 titer 20 and titer 100 plates.Following the incubation at 24 degrees Celsius, count the number of plaques formed in the 10 screening plates. To verify the putitive mutant plaques, core the plaque with a sterile wide bore pipette tip. Then transfer the plaque to sterile micro-centrifuge tube filled with 500 microliters of sterile SM buffer.Then incubate the micro-centrifuge tube at room temperature for at least two hours at room temperature. Next, mix one microliter of the cored phage solution with 200 microliters of the G1250 plating culture in a sterile round bottom tube. Then incubate the mixture at room temperature for 30 minutes.After 30 minutes, plate the cored phage solution in 2.5 milliliters of molten TB1 top agar and incubate at 24 degrees Celsius for 46 to 48 hours. Once the secondary plaques are visible, use a pipette tip to pick a single well-isolated Lambda cII mutant plaque. Transfer the plaque to a micro-centrifuge tube filled with 25 microliters of double distilled water in pipette several times.Leave the tube on boiling water for five minutes. Then centrifuge the tube at 18, 000 times G for three minutes at room temperature. Immediately after centrifugation, transfer 10 microliters of the supernatant to a new micro-centrifuge tube containing 40 microliters of the PCR master mix.After PCR amplification, purify the 432 base para-amplified product with the cII gene and its flanking regions incorporated using the PCR purification kit. Then sequence the cII transgene using a DNA analyzer to detect the mutations. The plaques obtained in both the titer 20 and the titer 100 plates are clearly visible by holding them next to a white light box and against a dark background.Here, the bar plot depicts the mutagenic potency of various chemicals in the physical compounds tested. This is done by analyzing the extent of increase in the relative cII mutant frequency isolated from the mouse embryonic fiber blasts. The bar plot depicts all these test reagents showing statistically significant fold increase in the cII mutant frequency.Next, the mutation spectra of the cII transgene in the mouse embryonic fiber blasts irradiated with UVB is demonstrated. The pie chart shows a significant increase in the relative frequency of single or tandem cytidine to thymidine transition at pyrimidine dinucleotides when compared to the control. Once mastered, this technique can be done in eight to ten hours if it is performed properly, excluding preparation times for bacterial streak plate in culturing.In DNA sequencing, subsequent to scoring the cII mutants. After watching this video, you should have a good understanding of how to perform mutation analysis of a test compound in an in vitro model system.

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Research

JoVE Journal

Immunology and Infection

The Lambda Select cII Mutation Detection System

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Сочетание Клей-ленты на основе выборки и флуоресценции На месте Гибридизация для быстрого обнаружения Сальмонеллы На Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Выявление инфекционных Касперского с поля, собранные Комары от Vero Пробирной культуры клеток

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

Выделение вариантов Fidelity РНК-содержащих вирусов и характеристика вирусов Частота мутаций

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

Упаковка с ВИЧ или FIV основе Lentivector Конструкции выражения и трансдукции VSV-G Pseudotyped вирусных частиц

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

После Cell-судьба<em&gt; E. палочки</em&gt; После заражения фагом лямбда

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Носовые салфетки для гриппа обнаружения вируса и изоляции от свиного

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Качественный и количественный анализы для обнаружения и определения характеристик белка Противомикробные

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Обнаружение энтерогеморрагические Escherichia Coli колонизации в мышиных принимающей системой неинвазивные в Vivo биолюминесценции

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

Обнаружение и удаление нуклеиназы загрязнения во время очистки рекомбинантных прототип пенистый вирус интеграза

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Высок объём платформы для проверки Salmonella spp. /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...