False-Positive Results and Contamination in Nucleic Acid Amplification Assays: Suggestions for a Prevent and Destroy Strategy

Borst, Annemarie; Box, A. T. A.; Fluit, Ad C.

doi:10.1007/s10096-004-1100-1

Cited by 266 publications

(189 citation statements)

References 77 publications

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“…Amplicon sizes were determined by comparison with a 100 bp DNA ladder (Invitrogen). Precautions to avoid contamination were followed, positive and negative controls were used in all reactions, and the procedure was reproduced several times 7 . Another protocol 27 that amplifies the capsid protein (C) was also performed to confirm the presence of DENV-1 and DENV-4 in the same sample (Fig.…”

mentioning

confidence: 99%

Co-Infection of Dengue Virus by Serotypes 1 and 4 in Patient From Medium Sized City From Brazil

Colombo

Vedovello

Reis

et al. 2013

Rev. Inst. Med. trop. S. Paulo

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mentioning

confidence: 99%

Co-Infection of Dengue Virus by Serotypes 1 and 4 in Patient From Medium Sized City From Brazil

Colombo

Vedovello

Reis

et al. 2013

Rev. Inst. Med. trop. S. Paulo

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“…There have been many reviews outlining the various procedures required to prevent and detect false positives, which include using separate rooms for different steps of the reaction, including negative controls at every PCR step, and regular decontamination of working areas with ultraviolet light or bleach. 7,10,37,38 Use of Formalin-Fixed Tissues for PCR Assays FFPE tissues can be used for PCR assays and 16S ribosomal RNA (rRNA) gene sequencing. However, since formalin treatment can fragment DNA, assays should be designed to work with smaller fragments.…”

Section: Quality Control In Molecular Diagnosismentioning

confidence: 99%

Nonculture Molecular Techniques for Diagnosis of Bacterial Disease in Animals

2014

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The past decade has seen remarkable technical advances in infectious disease diagnosis, and the pace of innovation is likely to continue. Many of these techniques are well suited to pathogen identification directly from pathologic or clinical samples, which is the focus of this review. Polymerase chain reaction (PCR) and gene sequencing are now routinely performed on frozen or fixed tissues for diagnosis of bacterial infections of animals. These assays are most useful for pathogens that are difficult to culture or identify phenotypically, when propagation poses a biosafety hazard, or when suitable fresh tissue is not available. Multiplex PCR assays, DNA microarrays, in situ hybridization, massive parallel DNA sequencing, microbiome profiling, molecular typing of pathogens, identification of antimicrobial resistance genes, and mass spectrometry are additional emerging technologies for the diagnosis of bacterial infections from pathologic and clinical samples in animals. These technical advances come, however, with 2 caveats. First, in the age of molecular diagnosis, quality control has become more important than ever to identify and control for the presence of inhibitors, cross-contamination, inadequate templates from diagnostic specimens, and other causes of erroneous microbial identifications. Second, the attraction of these technologic advances can obscure the reality that medical diagnoses cannot be made on the basis of molecular testing alone but instead through integrated consideration of clinical, pathologic, and laboratory findings. Proper validation of the method is required. It is critical that veterinary diagnosticians understand not only the value but also the limitations of these technical advances for routine diagnosis of infectious disease.Keywords bacterial infections, mass spectrometry, molecular diagnostic techniques, fluorescent in situ hybridization, quality control, realtime polymerase chain reaction Traditionally, pathogen identification for infectious disease was mostly based on isolation in culture, and this was also critical for fulfilling Koch's postulates. 24 Molecular identification methods have been used for infectious disease diagnosis since the 1980s, and ''molecular Koch's postulates'' were applied to bacterial pathogenicity at the gene rather than the wholeorganism level. 21,22 Molecular guidelines for establishing microbial disease causation were described as a reconsideration of Koch's postulates. 24 Among the nonculture methods, polymerase chain reaction (PCR) and DNA sequencing have been widely used to identify infectious pathogens. Recently, massive parallel DNA sequencing has rapidly developed for the identification of both described and new viral pathogens. The technology will likely revolutionize bacterial disease diagnosis in the near future. Not only have many nonculturable or difficult-to-culture bacterial pathogens become detectable, but many readily isolated bacterial infections can now be identified with nonculture methods. Molecular identification methods ca...

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“…It is unclear, however, how much of this increased falsepositive rate is attributable to the method itself versus higher rates of human error caused by a more complex and less frequently used test. 26 The decision of whether or not to perform viral NAT is left to the individual OPO. As of March 2008, 50% of OPOs performed HIV NAT for all donors, 25% performed it for a subset of donors or when requested by the transplant center, and 25% never performed HIV NAT.…”

Section: Defi Ning the Riskmentioning

confidence: 99%

High infectious risk organ donors in kidney transplantation: Risks, benefits, and current practices

Kucirka¹,

Dagher²,

Montgomery³

et al. 2010

Dialysis & Transplantation

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Approximately 9% of deceased kidney donors are classified by the Centers for Disease Control as high infectious risk donors (HRDs), donors thought to be at increased risk for having HIV infection. While the use of HRDs expands the organ supply, there is a small risk of infectious transmission. All donors are tested for antibodies to a variety of viral infections including HIV, hepatitis C virus (HCV), and hepatitis B virus; however, infections acquired in the weeks to months before death may not be serologically detectable, but will likely be transmitted to the recipient. Nucleic acid testing (NAT) shortens the window between acquisition of infection and serologic detectability, from approximately 22 days to 9 days for HIV and from 66 days to 7 days for HCV. Nucleic acid testing has not been universally adopted because it is expensive, time consuming, and has a higher rate of false positives compared with an enzyme‐linked immunosorbent assay (ELISA), which might lead to discarding viable organs. Further studies are needed to quantify the risk of infectious transmission from HRDs, identify patients on the waitlist who would most benefit from HRD receipt, and guide NAT policies.

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False-Positive Results and Contamination in Nucleic Acid Amplification Assays: Suggestions for a Prevent and Destroy Strategy

Cited by 266 publications

References 77 publications

Co-Infection of Dengue Virus by Serotypes 1 and 4 in Patient From Medium Sized City From Brazil

Co-Infection of Dengue Virus by Serotypes 1 and 4 in Patient From Medium Sized City From Brazil

Nonculture Molecular Techniques for Diagnosis of Bacterial Disease in Animals

High infectious risk organ donors in kidney transplantation: Risks, benefits, and current practices

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