Manual A Low-Cost Approach to PCR: Appropriate Transfer of Biomolecular Techniques

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After a min centrifugation at 10, rpm at infection with DV. A sim- Collection, Manassas, VA to identify the serotype. To confirm the utility 8. Of samples ramethylammonium chloride, 0.

The Complete Guide to PCR (How it Works, Primer Design, and Running Reactions) - Spider Silk Step 2

Of these latter samples, 2 demonstrated high titers of C for 30 sec, C for 1 min, and C for 2 min, ending IgM, and 2 contained high titers of IgG. The expected size of the amplified products ary As seen in lanes 4, 6, 9, 10, and 11, serotype 3 was as follows: basepairs for dengue-1, basepairs was the cause of the epidemic at this time. These positive for dengue-2, basepairs for dengue-3, and basepairs samples were later confirmed by viral isolation.

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During the for dengue Positive controls for the reverse transcriptase- month of July, the introduction of a new serotype dengue- PCR included RNA extracted from reference strains of each 2 was detected, as shown in Figure 1B and Table 1. In of the 4 serotypes and the plasmid pBD3L,5 which produces Figure 1B, the results of 3 dengue-3 positive samples are an amplicon of basepairs with primers D1 and TS3 and presented lanes 5, 6, and 11 and 2 dengue-2 positive sam- serves as an additional control for the amplification process.

Table 1 shows the temporal and geo- subtyping of dengue-2 and dengue-3 viruses was followed. Dengue-3 was Extraction of RNA was performed as described above, using the dominant serotype throughout the epidemic, while in the ml of supernatants of cells infected with previously iso- middle of the year dengue-2 was detected in the city of Ma- lated viral strains.

Each scriptase RAV-2 and 0. A new technique, RSS-PCR,20 was used to Reverse transcription and amplification were performed in a determine the subtypes of the dengue-2 and dengue-3 strains single tube as above, except with 30 cycles of amplification. Figure 2 lanes 1—4 shows Ten microliters of the products were analyzed by electro- the application of RSS-PCR to analyze dengue-2 subtypes.

Tissue culture flasks sents the endemic dengue-2 subtype in Latin America.

Lane 4 shows the result of the amplification of a 0. Lanes 6—9 by Gubler and others. This technique also requires that specimens be collected, trans- ported, and stored properly so as to ensure that the virus remains viable and the sample remains sterile. Over the last several years, RT-PCR has been applied to detection and typing of dengue virus,5,8—12 but until now, this tool has been used mostly on an experimental basis and not as a routine method in endemic countries.

It is important to point out that of the 8 discordant cases positive by RT-PCR and negative by viral isolation , 2 contained IgM antibodies and another 2 demonstrated high levels of IgG. Reverse transcriptase—polymerase chain reaction anal- which prevented its isolation but not its detection by RT- ysis of serum from patients suspected of dengue infection in Nica- PCR.

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The other 4 discordant cases probably resulted from ragua. A, samples from the beginning of the epidemic in January improper specimen transportation and storage, which com- , showing the presence of dengue Lane 1, negative reagent promised the ability of the virus to be isolated, rather than control water ; lane 2, negative amplification control water ; lane 3, negative extraction control water ; lane 4, ; lane 5, ; from false-positive PCR results, since all the necessary pre- lane 6, ; lane 7, ; lane 8, ; lane 9, ; lane 10, cautions were taken to avoid contamination of the samples ; lane 11, ; lane 12, ; lane 13, ; lane 14, during the PCR procedure24,25 and the results were repeated positive extraction control dengue-3 ; lane M, basepair [bp] several times on different days with identical results.

Thus, ladder Gibco—BRL, Gaithersburg, MD ; lane 16, positive amplifi- cation control dengue-1 ; lane 17, positive amplification control we believe that the RT-PCR is actually more sensitive than dengue-4 ; lane 18, positive amplification control dengue-3 ; lane viral isolation under these conditions, and equally specific. After 2 years gust , demonstrating the introduction of dengue Lane 1, neg- of relatively low incidence, the number of dengue cases ative reagent control water ; lane 2, negative amplification control water ; lane 3, negative extraction control water ; lane 4, ; confirmed by the detection of specific IgM antibodies in- lane 5, , lane 6, ; lane 7, ; lane 8, ; lane 9, creased abruptly in the beginning of This was unex- ; lane 10, ; lane 11, ; lane 12, ; lane 13, pected, since the dengue season usually begins later in the ; lane 14, positive extraction control dengue-3 ; lane M, year May and since a seroepidemiologic survey carried out bp ladder; lane 16, positive amplification control dengue-1 ; lane 17, positive amplification control dengue-4 ; lane 18, positive am- the previous year revealed a high level of dengue virus an- plification control dengue-3 ; lane 19, positive amplification control tibodies in the population de los Reyes J, Balmaseda A, dengue-2 ; lane 20, positive amplification control plasmid pBD3L.

As can be observed, the Nicaraguan dengue-3 demonstrated within 2 days that the serotype responsible was strains belong to RSS-PCR type C, which corresponds to in fact dengue-3 Figure 1A. These results were later con- group III of Lanciotti and others;16 all the Nicaraguan den- firmed by viral isolation. Note that the routinely during the entire year as part of the national system RSS-PCR patterns of Nicaraguan isolates of both dengue-2 of dengue viral surveillance; this allowed rapid typing of and dengue-3 appear to contain an extra amplified fragment samples from regions most affected by the epidemic Table that is not seen in the pattern of the control strains ; 1.

It also increased the quality of surveillance significantly basepairs for dengue-2 and ; basepairs for dengue In addition, a serotype that has not with the application of RT-PCR for detection and typing of circulated recently in a given population will encounter a dengue virus may be useful to other laboratories in the re- larger number of susceptible hosts, many of whom may have gion that present similar conditions or that cannot afford experienced a primary infection with a different dengue se- viral isolation or overseas processing of specimens to deter- rotype and may therefore be at increased risk for severe dis- mine the circulating dengue serotype s.

However, it is crit- ease, according to the sequential infection theory.

Introduction

The fact that dengue- Determination of the genotype of circulating dengue vi- 2 was detected in various regions of Nicaragua after mid- ruses is also important, since strains originating in Southeast suggested the possibility of dengue-2 circulation the Asia have been more often associated with severe dis- following year, when virus transmission begins again.

In ease.


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Na- tional health authorities were alerted immediately about the danger of the circulation of this viral strain. IN85 Rapid identification of in real time during an epidemic and to provide this infor- dengue virus serotypes by using polymerase chain reaction. J Clin Microbiol — Identification of dengue se- quences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood. Juan Jose Amador Director of the Luisa Amanda Rapid, single- Campos and Dr.

Alcides Gonzalez Director of the Centro Na- primers. J Virol Methods — We also thank Dr.

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We are grateful to Ana Maria Xet-Mull for Molecular epidemiology of dengue-1 assistance with translation. J Gen Virol — Arch Virol — Eva Harris, Infectious 65— Molecular evolution School of Public Health, University of California, Berkeley, and phylogeny of dengue-4 viruses. Virology — Rico-Hesse R, Molecular evolution and distribution of dengue viruses type 1 and 2 in nature. Virology — 1. Monath TP, Dengue: the risk to developed and developing Rapid subtyping of dengue viruses by restriction site- dengue hemorrhagic fever as a public health problem in the specific RSS -PCR.

Virology 86— Describes the theoretical basis of the technique, practical details, and the philosophy behind the technology transfer program that the author has developed over the past 10 years.

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A low-cost approach is outlined for use in the study of infectious diseases, which can be applied to other technologies and applications. This approach is especially useful for laboratories in developing countries, and in high school undergraduate and continuing education programs in the US. Appendices give information on construction of equipment, and contain sample charts and worksheets. Du kanske gillar. Spara som favorit. Skickas inom vardagar. Laddas ned direkt.