ASTM E2048-99(2006)
(Guide)Standard Guide for Detection of Nucleic Acids of the Mycobacterium Tuberculosis Complex and Other Pathogenic Mycobacteria by the Polymerase Chain Reaction Technique (Withdrawn 2014)
Standard Guide for Detection of Nucleic Acids of the Mycobacterium Tuberculosis Complex and Other Pathogenic Mycobacteria by the Polymerase Chain Reaction Technique (Withdrawn 2014)
SIGNIFICANCE AND USE
This guide is intended for use in any laboratory utilizing PCR or RT-PCR to amplify and detect nucleic acid sequences of mycobacteria from a biological preparation and to identify the species of origin.
The criteria used for the identification and evaluation of the amplification reactions should be administered by an individual trained in the use of molecular biological and microbiological techniques associated with PCR and MTB.
SCOPE
1.1 This guide covers basic considerations, criteria, principles and recommendations that should be helpful when developing, utilizing, or assessing PCR-specific protocols for the amplification and detection or identification of mycobacterial nucleic acids. This guide is not a specific protocol for the detection of specific mycobacteria. It is intended to provide information that will assist the user in obtaining high quality and reliable data. The guide is closely related to and should be used concurrently with the general PCR Guide E 1873.
1.2 This guide has been developed for use in any molecular biology or biotechnology laboratory. It may be useful for the detection of mycobacteria in clinical, diagnostic laboratories.
1.3 This guide does not cover details of the various methods such as gel electrophoresis that can be utilized to help identify PCR-amplified mycobacterial nucleic acid sequences, and it does not cover details of instrument calibration.
1.4 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.
1.5 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, and Biosafety Level 3 facilities are required for propagating and manipulating Mycobacteria tuberculosis cultures (). Safety guidelines should be adhered to according to NCCLS M29-T2, I17-P and other recommendations ().
WITHDRAWN RATIONALE
This guide covers basic considerations, criteria, principles and recommendations that should be helpful when developing, utilizing, or assessing PCR-specific protocols for the amplification and detection or identification of mycobacterial nucleic acids.
Formerly under the jurisdiction of Committee E55 on Manufacture of Pharmaceutical Products, this guide was withdrawn in August 2014. This standard was withdrawn without replacement due to its limited use by the industry.
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: E2048 − 99(Reapproved 2006)
Standard Guide for
Detection of Nucleic Acids of the Mycobacterium
Tuberculosis Complex and Other Pathogenic Mycobacteria
by the Polymerase Chain Reaction Technique
This standard is issued under the fixed designation E2048; 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 (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
This guide covers detection of nucleic acids (deoxyribonucleic acid (DNA) or ribonucleic acid
(RNA)) of mycobacteria and specific identification of the Mycobacterium tuberculosis complex
(MTBC,whichincludesM.tuberculosis,M.bovis,M.microtiandM.africunum),M.avium,M.leprae
and M. intracellulare nucleic acids by the polymerase chain reaction (PCR) technique.
PCR is a widely known molecular biology procedure that involves the amplification of a piece of
DNAby as much as a million-fold over the course of several hours. It is also possible to use PCR to
detect RNAby first copying RNAwith the enzyme reverse transcriptase to produce a complementary
DNA molecule, which is then amplified by PCR; the combined process is known as RT-PCR. The
amplified DNA fragments can then be detected, identified and quantitated by classical procedures of
biochemistry/molecular biology. As few as only several molecules of DNA in a biological test
specimen can be rapidly and accurately identified. PCR is used as a tool in molecular biology
laboratories for basic and applied research, in clinical laboratories to aid in the diagnosis of genetic,
neoplastic and infectious diseases, and in biotechnology laboratories for the preparation of biotech-
nology products and to test for contaminants.
Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis (TB) in humans, and TB
isoneoftheleadingcausesofhumandeathintheworld (1, 2) .Aboutonethirdofthepresentworld’s
population is infected with MTB (1). The definitive test for tuberculosis, isolation and specific
identification of MTB in culture, requires several weeks. Microscopic examination of acid-fast smears
is rapid, but non-specific and relatively insensitive. The value of using PCR or other DNA/RNA
amplification procedures for TB diagnosis is rapidity; clinical specimens can be evaluated within a
day. Thus, patient care and treatment can be initiated more rapidly when a specific diagnosis has been
determined.
This guide was developed byASTM in collaboration with DIN (German Institute for Standardiza-
tion) Subcommittee E3/E9 on Molecular Biological Detection of Mycobacteria, Department for
Medical Standards (NAMed). It is recommended that this mycobacteria-specific PCR guide be used
in conjunction withASTM’s general PCR Guide E1873. The combination of the two guides provides
recommendations, basic considerations, criteria, and principles that should be employed when
developing, utilizing or assessing PCR-specific protocols for the detection of the DNA or RNA of
specific mycobacteria.
This guide assumes a basic knowledge of microbiology and molecular biology. It assumes the
availability of, and the ability to search the literature for, mycobacteria target-specific PCR protocols.
1. Scope
1.1 This guide covers basic considerations, criteria, prin-
This guide is under the jurisdiction of ASTM Committee E55 on Manufacture
ciples and recommendations that should be helpful when
of Pharmaceutical Products and is the direct responsibility of Subcommittee E55.04
on General Biopharmaceutical Standards.
Current edition approved Feb. 1, 2006. Published February 2006. Originally
approved in 1999. Last previous edition approved in 1999 as E2048 – 99. DOI: The boldface numbers in parentheses refer to the list of references at the end of
10.1520/E2048-99R06. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2048 − 99 (2006)
developing, utilizing, or assessing PCR-specific protocols for similar length and base composition to the target gene se-
the amplification and detection or identification of mycobacte- quence, and that contains primer binding regions identical to
rial nucleic acids. This guide is not a specific protocol for the those of the target sequence.
detection of specific mycobacteria. It is intended to provide
3.2.2 restriction enzymes, n—naturally occurring proteins
information that will assist the user in obtaining high quality
(also called restriction endonucleases) that are purified from
and reliable data. The guide is closely related to and should be
bacteria and that recognize specific nucleic acid sequence
used concurrently with the general PCR Guide E1873.
patterns (sites) and cleave the nucleic acid at or near that
sequence (site).
1.2 This guide has been developed for use in any molecular
biology or biotechnology laboratory. It may be useful for the
4. Significance and Use
detection of mycobacteria in clinical, diagnostic laboratories.
4.1 This guide is intended for use in any laboratory utilizing
1.3 This guide does not cover details of the various methods
PCR or RT-PCR to amplify and detect nucleic acid sequences
such as gel electrophoresis that can be utilized to help identify
of mycobacteria from a biological preparation and to identify
PCR-amplified mycobacterial nucleic acid sequences, and it
the species of origin.
does not cover details of instrument calibration.
4.2 The criteria used for the identification and evaluation of
1.4 Thisguidedoesnotcoverspecificvariationsofthebasic
the amplification reactions should be administered by an
PCR or RT-PCR technology (for example, quantitative PCR,
individual trained in the use of molecular biological and
multiplex PCR and in situ PCR), and it does not cover details
microbiological techniques associated with PCR and MTB.
of instrument calibration.
1.5 Warning—Laboratory work involving certain clinical
5. Background Information about TB, MTB, Other
specimens and microorganisms can be hazardous to personnel.
Mycobacteria, and Detection of Mycobacteria by PCR
Precaution: Biosafety Level 2 facilities are recommended for
5.1 The mycobacteria are acid-fast, non-motile, rod-shaped,
potentiallyhazardouswork,andBiosafetyLevel3facilitiesare
aerobic bacteria that do not form spores. They contain several
required for propagating and manipulating Mycobacteria tu-
species pathogenic to humans. The primary human pathogens
berculosis cultures (3). Safety guidelines should be adhered to
are members of the Mycobacterium tuberculosis complex (M.
according to NCCLS M29-T2, I17-P and other recommenda-
tuberculosis, M. bovis, M. africanum and M. microti) and M.
tions (3).
leprae. Other mycobacteria, such as the M. avium complex (M.
avium and M. intracellulare ) and M. kansasii, cause disease in
2. Referenced Documents
3 immunocompromised individuals.
2.1 ASTM Standards:
5.2 Tuberculosis has been a major public health problem in
E1873 Guide for Detection of Nucleic Acid Sequences by
the Polymerase Chain Reaction Technique the world for many centuries, particularly in overcrowded city
areas. In the 18th and 19th centuries, TB was responsible for a
2.2 NCCLS Standards:
quarter of all adult human deaths in European cities (4).
M29-T2 Protection of Laboratory Workers from Infectious
Severalpublichealthmeasuresthatfinallyledtoaconsiderable
Disease Transmitted by Blood, Body Fluids, and Tissue-
improvement include the pasteurization of milk, and the
Second Edition; Tentative Guideline
antibiotics streptomycin (introduced in 1944) and
C24-A2 Statistical Quality Control for Quantitative Mea-
p-aminosalicylic acid (introduced in 1946) (4). However, the
surements: Principles and Definitions; Approved
emergenceofdrug-resistantMTBstrainsisbecomingaserious
Guideline-Second Edition
problem. For this and other reasons,TB is presently on the rise
MM3-A Molecular Diagnostic Methods for Infectious Dis-
again and is one of the leading infectious causes of death of
eases; Approved Guideline
adult humans in the world.
3. Terminology 5.2.1 The isolation of a species from the MTBC is required
forthedefinitivediagnosisoftuberculosis.Routineculturesare
3.1 Basic PCR definitions apply according to the general
time-consuming and can take up to eight weeks. Microscopic
PCR Guide E1873 (Section 3).
examination of acid-fast smears is the most rapid method for
3.2 Definitions of Terms Specific to This Standard:
the detection of mycobacteria, but it is insensitive and non-
3.2.1 internal control, n—in PCR, a control used to assess
specific.Immunologicalandserologicaltechniquesarelimited,
the amplifiability of the reaction, that is, to determine whether
in general, due to poor sensitivity or specificity, or both (5, 6).
or not PCR inhibitors may be present in the reaction; for
Species-specific nucleic acid probes have significantly im-
example, an internal control can be a synthetic DNA segment
proved the opportunity for rapid confirmation of culture results
that can be added to the sample prior to amplification, that is of
for several mycobacterial species (7).
5.3 The M. avium complex (MAC) consist of 28 serovars
(subspecies) of two distinct species, M. avium and M. intrac-
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
ellulare (8). The criteria used to distinguish M. avium from M.
Standards volume information, refer to the standard’s Document Summary page on
intracellularearenowwellestablished (9-11).MACorganisms
the ASTM website.
are ubiquitous in nature and have been isolated from water,
Available from the National Committee for Clinical Laboratory Standards, 771
E. Lancaster Ave., Villanova, PA 19085. soil, plants, and other environmental sources (12). These
E2048 − 99 (2006)
organisms are of low pathogenicity and frequently colonize specimens. Because the volume of specimen used to inoculate
healthy individuals without causing disease. Person-to-person cultures is usually much greater than the volume tested by
transmission is thought to be unlikely, but many investigators PCR, mycobacteria may be delivered to the culture, but not to
believe that pulmonary disease may result from inhalation of the PCR reaction.
infectious environmental aerosols. MAC infections have be- 5.5.2 Clinical studies have shown that PCR assays can
come increasingly more common in the United States, and M. detect MAC directly in respiratory specimens and in blood
avium is the most common non-tuberculous mycobacterial (23-27).Manyoftheseassayscandistinguishwhichofthetwo
speciesassociatedwithhumandisease.Thegreatestincreasein MAC species, M. avium or M. intracellulare, is present in the
MAC infections during the past decade has been in AIDS specimen. The specificity of these tests ranges from 97 to
patients for whom MAC has become the third most common 100 %. Furthermore, these MAC-specific tests do not cross-
opportunistic disease (13). In one report, 98 % of MAC react with other mycobacteria. Clinical specimens that yielded
infections in 45AIDS patients were due to M. avium, whereas M. tuberculosis, or other non-MAC mycobacteria, when cul-
40 % of MAC infections in patients withoutAIDS were due to tured gave negative results when tested by MAC-specific PCR
M. intracellulare (14). assays. Sensitivity for MAC is generally greater than 90 %.
5.3.1 The isolation of MAC by culture is required for
6. Principle of the PCR Method
definitive diagnosis of MAC infection. However, routine cul-
tures are time consuming and can take up to eight weeks for
6.1 See Guide E1873 (Section 5) for a description of the
final diagnosis. Additionally, culture does not distinguish
PCR method.
between M. avium and M. intracellulare infection. Several of
6.2 In addition to 6.1, the amplification of target sequences
the therapeutic agents used currently in the treatment of MAC
takes place in vitro. Procedures for detection of specific
disease have different in vitro activity against M. avium and M.
amplified products that allow a differentiation to be made
intracellulare (15). As additional therapeutic agents become
between species of the MTBC and ubiquitous mycobacterial
available, the ability to distinguish between M. avium and M.
species should be used preferentially.
intracellulare may become more important.
5.4 Mycobacterium leprae, the causative agent of leprosy, 7. Target Material
remains a serious health problem worldwide (16). Diagnosis of
7.1 For general information see Guide E1873 (Sections 6
M. leprae infections is a problem due to the fact that this
and 9).
organism cannot be cultured by conventional methods. Classi-
7.2 In addition to 7.1, for the detection of nucleic acid
cal methods of diagnosis, such as microscopic examination of
sequences of the MTBC, immediate transport of the test
skin biopsies and antibody testing lack sensitivity and speci-
material to the laboratory is recommended. It is possible that
ficity (17). However, PCR can be used for rapid and sensitive
transport periods of >48 h can interfere with the nucleic acid
diagnosis of M. leprae infections.
detection.
5.5 PCR has proven to be a useful procedure for the
7.3 Typical biological specimens for the detection of myco-
detection of mycobacteria and the specific identification of the
bacteria or their nucleic acid sequences include:
species present. Mycobacterial DNA in a clinical sample is
7.3.1 Specimens Used in Biotechnology or Basic Molecular
typically extracted and amplified, and the PCR product is then
Biology Research Laboratories—Cultures of mycobacteria,
identified. Depending on the PCR amplification target em-
purified preparations of mycobacteria or mycobacterial nucleic
ployed, the analytic sensitivity of amplification assays ranges
acid.
from about 1 to 100 mycobacteria (17). Primers used in the
7.3.2 Specimens Used in Clinical, Diagnostic Laboratories
PCR amplification from mycobacterial genomes can be either
in the Case of Suspected MTB Infection—Respiratory sample
species-specific or genus-specific. PCR-based tests specific for
material (for example, sputum, bronchoalveolar lavages), gas-
mycobacteria have been shown to improve the rapid diagnosis
tric juice, stomach aspirate, urine, sperm and prostatic secre-
of tuberculosis and other mycobacteria-caused diseases by
tion, puncture exudate, cerebrospinal fluid, bone marrow and
allowing the direct detection of mycobacteria in clinical
biopsy material (tissues).
specimens.
5.5.1 Clinical studies have demonstrated that PCR-based 7.4 Methods currently used for extraction of the target
assays accurately detect MTB
...
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