Multiplex Allele-Specific Amplification from Whole Blood for Detecting Multiple Polymorphisms Simultaneously

Zhu, Jianjie; Chen, Lanxin; Mao, Yong; Zhou, Huan; Li, Rui; Wang, Weipeng

doi:10.1089/gtmb.2012.0261

Cited by 3 publications

(2 citation statements)

References 24 publications

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“…18 Hypotonic solution, increased pH, and MgCl 2 concentration have also been successfully used in the genotypic identification of closely related alleles. 19 The addition of anticoagulants was shown to be helpful in the amplification of human hemochromatosis and apoE genes. Na-heparin, a commonly used anticoagulant for the collection of blood from humans, was cited to be better than using either EDTA or sodium citrate.…”

Section: Resultsmentioning

confidence: 99%

Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification

et al. 2016

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Malaria remains one of the most prevalent infectious diseases and results in significant mortality. Isothermal amplification (loop-mediated isothermal amplification) is used to detect malarial DNA at levels of ~1 parasite/µL blood in ≤30 minutes without the isolation of parasite nucleic acid from subject’s blood or saliva. The technique targets the mitochondrial cytochrome oxidase subunit 1 gene and is capable of distinguishing Plasmodium falciparum from Plasmodium vivax. Malarial diagnosis by the gold standard microscopic examination of blood smears is generally carried out only after moderate-to-severe symptoms appear. Rapid diagnostic antigen tests are available but generally require infection levels in the range of 200–2,000 parasites/µL for a positive diagnosis and cannot distinguish if the disease has been cleared due to the persistence of circulating antigen. This study describes a rapid and simple molecular assay to detect malarial genes directly from whole blood or saliva without DNA isolation.

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Section: Resultsmentioning

confidence: 99%

Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification

et al. 2016

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“…However, the recovery rate of chromosomal DNA eluted from bacterial cells with this direct qPCR master mix remains unknown18192021. The soft membranes of mammalian cells easily burst under hyperosmolarity or hypoosmolarity, thus allowing intermediate direct qPCR elongation through the additional boiling of crude DNA extractions2324. However, unlike mammalian cells, prokaryotic cells have solid cell walls1025.…”

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confidence: 99%

A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells

Soejima

Xiao

Abe

2016

Sci Rep

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Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 100 cfu/ml for the test sample compared with a detection limit of 1.6 × 103 cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research.

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A Novel Direct PCR Lysis Buffer Can Improve PCR from Meat Matrices

et al. 2018

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Multiplex Allele-Specific Amplification from Whole Blood for Detecting Multiple Polymorphisms Simultaneously

Cited by 3 publications

References 24 publications

Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification

Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification

A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells

A Novel Direct PCR Lysis Buffer Can Improve PCR from Meat Matrices

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