ASTM E1873-97

Standard Guide for Detection of Nucleic Acid Sequences by the Polymerase Chain Reaction Technique

SCOPE
1.1 This guide covers guidelines, recommendations, basic considerations, criteria, and principles to be employed when developing, utilizing, or assessing PCR procedures and specific protocols for the amplification and detection of nucleic acid sequences. This guide is not intended to be a standard procedure with a list of requirements for PCR detection of nucleic acids. This guide is intended to provide information that will assist the user in obtaining quality and reliable data.  
1.2 Nucleic acid sequences that can be amplified by PCR include RNA, as well as DNA sequences; RNA sequences are suitable targets for PCR following reverse transcription of the RNA to complementary DNA (cDNA). This type of amplification technique is often called reverse transcription-PCR (RT-PCR).  
1.3 This guide has been developed for use in any molecular biology/biotechnology laboratory. This includes, but is not limited to, laboratories that specializein the diagnosis of human, animal, plant, or bacterial diseases.  
1.5 This guide is a general one; it does not cover the additional guidance that would be needed for specific applications, for example, for the PCR detection of nucleic acid sequences of specific microorganisms.  
1.6 This guide does not cover details of the various methods that can be utilized to identify PCR-amplified DNA sequences.  
1.7 This guide does not cover specific variations of the basic PCR or RT-PCR technology (for example, quantitative PCR, multiplex PCR, and in-situ PCR), and it does not cover details of instrument calibration.  
1.8 Warning--Laboratory work involving certain clinical specimens and microorganisms can be hazardous to personnel. Precaution-- Biosafety level 2 facilities are recommended for potentially hazardous work. Safety guidelines should be adhered to in accordance with NCCLS M29-T2, I17-P, and other recommendations.

General Information

• Status: Historical
• Publication Date: 31-Dec-1996
• Technical Committee: E48 - Bioenergy and Industrial Chemicals from Biomass

Relations

• Replaced By: ASTM E1873-97(2002) - Standard Guide for Detection of Nucleic Acid Sequences by the Polymerase Chain Reaction Technique
• Effective Date: 10-Apr-1997
• Referred By: ASTM E2800-11(2017) - Standard Practice for Characterization of <emph type="ital">Bacillus</emph> Spore Suspensions for Reference Materials
• Effective Date: 01-Jan-1997

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
Designation: E 1873 – 97
Standard Guide for
Detection of Nucleic Acid Sequences by the Polymerase
Chain Reaction Technique
This standard is issued under the fixed designation E 1873; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) 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 and for DNA sequencing. There is an abundance of publications
addressing laboratory procedures and specific protocols for various applications. The field of PCR is
advancing so rapidly; however, it is necessary to frequently modify and update these procedures and
specific protocols. Rather than describe a standard procedure and list requirements that would
probably soon become outdated, this guide consists of guidelines, recommendations, basic consider-
ations, 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
Biological Systems in collaboration with DIN (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 microbiology and molecular biology. It assumes the
availability of basic references in PCR for general procedures (see Refs 1-7) and the ability to search
the literature for target-specific protocols.
1. Scope limited to, laboratories that specialize in the diagnosis of
human, animal, plant, or bacterial diseases.
1.1 This guide covers guidelines, recommendations, basic
1.4 This guide conveys the general procedural terminology
considerations, criteria, and principles to be employed when
of PCR technology used for the detection of nucleic acids.
developing, utilizing, or assessing PCR procedures and specific
1.5 This guide is a general one; it does not cover the
protocols for the amplification and detection of nucleic acid
additional guidance that would be needed for specific applica-
sequences. This guide is not intended to be a standard
tions, for example, for the PCR detection of nucleic acid
procedure with a list of requirements for PCR detection of
sequences of specific microorganisms.
nucleic acids. This guide is intended to provide information
1.6 This guide does not cover details of the various methods
that will assist the user in obtaining quality and reliable data.
that can be utilized to identify PCR-amplified DNA sequences.
1.2 Nucleic acid sequences that can be amplified by PCR
1.7 This guide does not cover specific variations of the basic
include RNA, as well as DNA sequences; RNA sequences are
PCR or RT-PCR technology (for example, quantitative PCR,
suitable targets for PCR following reverse transcription of the
multiplex PCR, and in situ PCR), and it does not cover details
RNA to complementary DNA (cDNA). This type of amplifi-
of instrument calibration.
cation technique is often called reverse transcription-PCR
1.8 Warning—Laboratory work involving certain clinical
(RT-PCR).
specimens and microorganisms can be hazardous to personnel.
1.3 This guide has been developed for use in any molecular
Precaution—Biosafety level 2 facilities are recommended for
biology/biotechnology laboratory. This includes, but is not
potentially hazardous work (8). Safety guidelines should be
adhered to in accordance with NCCLS M29-T2, I17-P, and
other recommendations (8).
This guide is under the jurisdiction of ASTM Committee E-48 on Biotechnol-
ogy and is the direct responsibility of Subcommittee E48.02 on Characterization and
Identification of Biological Systems. 2. Referenced Documents
Current edition approved April 10, 1997. Published June 1997.
2 2.1 NCCLS Standards:
The boldface numbers in parentheses refer to the list of references at the end of
I17-P Protection of Laboratory Workers form Instrument
this standard.
Copyright © ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
E 1873
Biohazards; Proposed Guideline (1991) and precision of the amplification reaction. There are several
C24-A Internal Quality Control Testing: Principles and ways to achieve hot-start PCR. All methods involve withhold-
++
Definitions; Approved Guideline (1991) ing a critical component (for example, polymerase or Mg )
M29-T2 Protection of Laboratory Workers from Infectious during the reaction setup at room temperature. Reaction tubes
Disease Transmitted by Blood, Body Fluids, and Tissue— are then heated to a temperature exceeding 60°C, at which
Second Edition; Tentative Guideline (1991) point the critical component is either added to the tube, comes
GP5-A Clinical Laboratory Waste Management; Approved in contact with the reaction mixture (for example, a hot start
Guideline (1993) wax), or is activated (for example, a DNA polymerase used for
MM3-A Molecular Diagnostic Methods for Infectious Dis- hot start PCR).
eases; Approved Guideline (1995)
3.2.9 hybridization, n—the specific annealing of a comple-
mentary DNA strand (for example, an oligonucleotide DNA
3. Terminology
primer) to the target DNA or RNA or amplified DNA segment
3.1 Basic concepts for quality assurance in the laboratory in a medium containing an appropriate buffer composition, pH
should be followed, in accordance with NCCLS C24-A.
value, and temperature range.
3.2 Definitions of Terms Specific to This Standard:
3.2.10 nested PCR, n—a modification of PCR that uses
3.2.1 annealing, v—in PCR, the binding or hybridization of
nested sets of PCR primers to enhance the sensitivity and
two complementary strands of DNA or of DNA and RNA, as
specificity of the reaction. A nested PCR protocol consists of
in the hybridization of oligonucleotide primers with DNA or
two rounds of PCR amplification and utilizes two sets of
RNA.
primer pairs. In the first round of amplification, the outermost
3.2.2 carryover contamination, n—in PCR, the adulteration
primer pair is used to generate an amplification product that is
of a PCR with amplified material (PCR products) from a
then subjected to a second round of amplification in the
previously amplified sample or with nucleic acids from another
presence of the internal primer pair.
source. This potentially can cause a false positive result.
3.2.11 polymerase chain reaction, PCR, n—an in vitro
3.2.3 chaotrope, n—in PCR, a chemical denaturing agent
laboratory method for the enzymatic amplification of nucleic
that lowers the melting temperature of double-stranded nucleic
acid sequences (1-3). Two DNA oligonucleotide primers an-
acids.
neal with their complementary DNA strands and flank (that is,
3.2.4 denaturation, n—in PCR, separation of double-
border) the segment to be amplified. The increase in amount
stranded DNA into single-stranded DNA. This can be accom-
(amplification) of the DNA segments occurs during repeated
plished with heat, extremes of pH, or exposure to certain
cycles consisting of three steps: heat denaturation of the
chemicals such as chaotropic agents, plus heat, usually.
double-stranded DNA, cooling to effect annealing of the
3.2.5 DNA polymerase, n—for PCR, usually a thermostable
primers to their complementary DNA strands, and enzymatic
enzyme used in PCR that catalyzes the repeated synthesis of
extension of the annealed primers by DNA polymerase at an
DNA under the selected reaction conditions of the method. The
optimal temperature. The amplification results in a near expo-
PCR, however, also can be performed with DNA polymerases
nential increase in the amount of the nucleic acid target defined
that are not thermostable, if fresh enzyme is added before each
by the primers.
cycle.
3.2.12 PCR buffer, n—a liquid medium that provides the
3.2.6 DNA probe, n—for PCR or RT-PCR, an oligonucle-
appropriate salts, cofactors, and pH value required for ampli-
otide that is complementary to a portion of the amplified DNA
fication of DNA by PCR.
product, contains a sufficient number of nucleotides, generally
3.2.13 PCR product, n—the amplified DNA synthesized by
20 to 35, to facilitate specific hybridization with the amplified
DNA polymerase in a PCR. This term is sometimes called
DNA product, and may be labeled with a detection molecule.
amplicon.
It is common to use probes that can bind to the amplified DNA
3.2.14 primer, n—for PCR, an oligonucleotide of defined
product at positions located between annealing sites of PCR
length, generally 20 to 30 bases, complementary to one strand
primers. In some procedures, however, the probe can be used
of DNA from the target sequence of interest. A primer pair
as the PCR primer.
defines the segment of the target DNA to be amplified. Primers
3.2.7 extension (of primer), n—in PCR, the synthesis of a
can be labeled. For RT-PCR, a primer can be an oligonucle-
new strand of DNA that is complementary to the target DNA,
otide of defined length complementary to an RNA target
used as a template, by the addition of nucleotide triphosphates
sequence of interest, a random hexamer or oligo deoxythymi-
to the oligonucleotide primer annealed to the target DNA.
dine.
Extension is catalyzed by an enzyme with DNA polymerase
3.2.15 primer-dimer, n—in PCR, the extension and ampli-
activity.
fication of primer pairs (independent of target DNA), usually
3.2.8 hot-start PCR, n—a variation of PCR designed to
caused when there is some complementarity between the two
minimize the formation of non-specific amplification products,
primers at the 38 ends. These form products that are usually the
often exhibited by smearing on gels, that may occur during the
length of the total of the two primers minus the overlap. Since
reaction setup, thereby enhancing the specificity, sensitivity,
they are relatively small and are complementary to the free
primers, they can amplify efficiently and compete with and
hinder amplification of the target DNA in PCR.
Available from the National Committee for Clinical Laboratory Standards, 771
E. Lancaster Avenue, Villanova, PA 19085. 3.2.16 reaction tubes, n—for PCR, tubes that can be heated
NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
E 1873
to 100°C and cooled to 4°C without any damage resulting in sequences. The increase in amount (amplification) of target
leakage of contents. They should allow a rapid and homoge- DNA occurs by an enzyme with DNA polymerase activity in
neous transfer of heat from the instrument heating block to the the presence of two, or more, oligonucleotide primers, deoxy-
++
reaction mix to occur during the PCR procedure. They should nucleoside triphosphates, Mg and a defined reaction buffer.
not be made with substances known to be inhibitory to PCR Amplification of the DNA follows a reaction cycle that consists
(2,9,10). of the following.
3.2.17 reverse transcription–PCR, RT-PCR, n—an in vitro
5.1.1 Heating the reaction mixture to a temperature suffi-
laboratory method for using RNA as the target molecule for
cient to denature the two strands of the target DNA.
amplification (3,11). A cDNA copy of the RNA target is made
5.1.2 Cooling the reaction mixture to a temperature that
by annealing a complementary DNA oligonucleotide to the
allows the primers to anneal with the target DNA strands.
RNA target followed by the enzymatic extension of the
5.1.3 Controlling at a temperature that allows extension of
annealed primer by an enzyme with reverse transcriptase
the annealed primers by DNA polymerase to occur on both
activity. The cDNA:RNA hybrid is then heat denatured and a
single strands.
complementary primer anneals to the cDNA, and a second
5.1.4 The preceding steps (5.1.1-5.1.3) constitute a cycle.
DNA strand is synthesized by an enzyme with DNA poly-
The cycle is repeated, (for example, 30 times), providing for
merase activity. The increase in amount (amplification) of the
repeat amplification of the target and any amplified material
cDNA segments occurs during repeated cycles of heat dena-
produced in the previous cycles. Some amplification protocols
turation of the double-stranded DNA, annealing of the primers
lack a discrete extension step, and extension occurs while the
to their complementary DNA strands, and enzymatic extension
reaction mixture is being heated or cooled between the
of the annealed primers by DNA polymerase (see 3.2.11). The
annealing temperature and the denaturation temperature.
amplification (of DNA) results in a near exponential increase in
5.2 The RT-PCR is an in vitro method that enables the
the amount of the original RNA segment defined by the
amplification and subsequent detection of cDNA reverse-
antisense primer.
transcribed from segments of RNA. The replication of RNA
3.2.18 reverse transcription/polymerase enzyme, n—for RT-
segments occurs in two steps. A cDNA copy of the original
PCR, a single enzyme that mediates reverse transcription of
RNA target is made by an enzyme with reverse transcriptase
RNA to cDNA as well as amplification of cDNA by PCR. The
activity in the presence of a reverse transcription oligonucle-
enzyme rTth is an example of a thermostable DNA polymerase
otide primer, deoxynucleoside triphosphates, a divalentcation
that can be used to reverse transcribe RNA efficiently in the
and a defined reaction buffer. In the second step the amount of
++
presence of Mn at elevated temperature, and then subse-
cDNA is increased (amplified) by PCR. The increase in amount
quently act as a DNA polymerase to amplify the cDNA target
(amplification) of cDNA is catalyzed by an enzyme with DNA
in a single tube. Alternatively, two enzymes may be used, one
polymerase activity in the presence of two oligonucleotide
a reverse transcriptase which transcribes the RNA to cDNA, ++
primers, deoxynucleoside triphosphates, Mg , and a defined
such as M-MLV or AMV, and the other, a DNA polymerase
reaction buffer. Amplification of the RNA follows a reaction
that amplifies the cDNA by PCR.
cycle that consists of the following.
3.2.19 target
...