ASTM E1873-06

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Designation: E1873 − 06
StandardGuide for
Detection of Nucleic Acid Sequences by the Polymerase
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Chain Reaction Technique
This standard is issued under the fixed designation E1873; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
This guide applies to the detection of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)
sequences by the polymerase chain reaction (PCR) technique. The PCR is used as a tool in many
molecular biology laboratory settings and for diverse reasons, for example, for amplification and
detection of nucleic acid sequences. There is an abundance of publications addressing laboratory
procedures and specific protocols for various applications. The field of PCR is advancing so rapidly
thatitisnecessarytofrequentlymodifyandupdatetheseproceduresandspecificprotocols.Thisguide
consists of guidelines, recommendations, basic considerations, criteria, and principles that should be
employed when developing, utilizing, or assessing PCR procedures and specific protocols for the
amplification and detection of nucleic acid sequences.
This guide was developed by Subcommittee E48.02 on Characterization and Identification of
BiologicalSystems.The1997editionofthisguidewasdevelopedincollaborationwithDIN(German
Institute for Standardization) Committee E9 on Serodiagnosis of Infectious Diseases and Diseases of
the Immune System, Department for Medical Standards (NAMed).
This guide assumes a basic knowledge of molecular biology. It assumes the availability of basic
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references in PCR for general procedures (see Refs 1-7) and the ability to search the literature for
target-specific protocols.
1. Scope 1.3 This guide has been developed for use in any molecular
biology/biotechnology laboratory. This includes, but is not
1.1 This guide covers guidelines, recommendations, basic
limited to, laboratories that specialize in the diagnosis of
considerations, criteria, and principles to be employed when
human, animal, plant, or bacterial diseases.
developing,utilizing,orassessingPCRproceduresandspecific
1.4 This guide conveys the general procedural terminology
protocols for the amplification and detection of nucleic acid
of PCR technology used for the detection of nucleic acids.
sequences. This guide is not intended to be a standard
procedure with a list of requirements for PCR detection of
1.5 This guide is general; it does not cover the additional
nucleic acids. This guide is intended to provide information
guidance that would be needed for specific applications, for
that will assist the user in obtaining quality and reliable data.
example, for the PCR detection of nucleic acid sequences of
specific microorganisms.
1.2 Nucleic acid targets for PCR include DNA, as well as
RNA ; RNA sequences are suitable targets for PCR following
1.6 Thisguidedoesnotcoverdetailsofthevariousmethods
reverse transcription of the RNA to complementary DNA
that can be utilized to identify PCR-amplified DNAsequences.
(cDNA). This type of amplification technique is known as
1.7 Thisguidedoesnotcoverspecificvariationsofthebasic
reverse transcription-PCR (RT-PCR).
PCR or RT-PCR technology (for example, quantitative PCR,
real-time PCR, multiplex PCR, and in situ PCR), and it does
not cover details of instrument calibration.
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This guide is under the jurisdiction ofASTM Committee E55 on Manufacture
1.8 Warning—Laboratory work involving certain clinical
of Pharmaceutical Products and is the direct responsibility of Subcommittee E55.04
specimens and microorganisms can be hazardous to personnel.
on General Biopharmaceutical Standards.
Current edition approved Nov. 1, 2006. Published November 2006. Originally
Warning—Biosafety level 2 (or higher) facilities are recom-
approved in 1997. Last previous edition approved in 2004 as E1873–04. DOI:
mended for biohazard work (8). Safety guidelines should be
10.1520/E1873-06.
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adhered to in accordance with CLSI M29-A2 and other
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this standard. recommendations (8).
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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E1873 − 06
2. Referenced Documents 3.1.9 hot-start PCR, n—a variation of PCR designed to
minimize the formation of non-specific amplification products,
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2.1 CLSI Standards:
often exhibited as smearing on electrophoretic gels, that may
C24-A2Statistical Quality Control for Quantitative Mea-
occur during the reaction setup, thereby enhancing the speci-
surements: Principles and Definitions; Approved
ficity, se
...