28 August 2006 [Edited]
Contact: Stephen M. Apatow, Director of
Research and Development, Humanitarian University
Consortium GraduateStudies
Center
for Medicine,
Veterinary
Medicine and Law. Email: s.m.apatow@pathobiologics.org
IDIN Communication: Biodefense Threat Analysis &
Communications Center
A new IHR
level precedent is being set for the surveillance of pathogens circulating in UN member countries that present
a international security threat.
From a risk management standpoint, the present threat to the international
community is proportional to the lack of a clear picture as to where pathogens
are evolving in both human and animal populations. If countries cannot
be trusted or do not have the infrastructure to conduct surveillance activities,
substantive data (tests) must be collected and analyzed, so that appropriate
response plans can be implemented for containment and control. That said,
our understanding of an immediate threat (such as a highly pathogenic human
to human transmissible strain of H5N1 - Immediate
IHR implementation), might help us in a decision making process to restrict international
travel/exposure to the human transmission vehicle, but does little
to facilitate progress in the globalization experiment. So in short, our
capacity to optimize infrastructure (surveillance, containment and control
strategies) would be a logical goal and one that I believe would be supported
by UN member countries.
Dear Colleagues:
As per the progress, development and need for integration of technologies
highlighted at the recent conference "Systems Integration in
Biodefense: Building a Blueprint for Policy and Preparedness," significant
challenges still remain in regards to licensing and field validation.
Exhibits/Sponsors
included the CombiMatrix group that recently launched the new CustomArray(TM)
90K (Genetic
Engineering News, 9 May 2006), a high-density, customizable, re-usable
microarray with over 94,000 unique DNA probes facilitating whole-genome
gene expression, SNP genotyping, comparative genomic hybridization (CGH),
tiling, ChIP-on-chip, and resequencing.
[The 392,000 array being developed at Livermore Microarray Center (LMAC) expands the microarray discussion to a new level.]
Another initiative under development was presented Hong Cai, Ph.D.,
Technical Staff Member, Bioscience Division, Los Alamos National Laboratory:
The Nucleic Acid Dipstick for Rapid Field Pathogen Detection is
a rapid, sensitive, inexpensive (<$10/assay), and easy-to-operate nucleic
acid-based dipstick device (of the size of ball point pen, like those in
home pregnancy test strips sold in stores) to detect and distinguish multiple
pathogens in 60 minutes (including the sample handling, NA extraction, amplification
and visualization). This presentation included data to demonstrate rapid
sensitive detection of as little as several copies of bacillus anthracis DNA
using isothermal amplification and nucleic acid dipstick assay.
Multiple amplification primer pairs are used to amplify multiple pathogen
targets, and multiple labeling (blue lmicrospheres conjugated with multiple
labeling sequences complimentary to different pathogen sequences) and
capture oligomer probes (immobilized onto lateral flow membrane) to capture
them onto different sports onto dipstick. The method is good for multiplexed
detection of about 10 different pathogens in one isothermal amplification
assay chamber. If there is a need to amplify more than 10 pathogens, eg.
100 pathogens, several isothermal amplification chambers can be bundled into
one dipstick device, and then captured onto a membrane spotted with 100 spots
for eye visualization.
An Inexpensive
and Simple Nucleic Acid Dipstick for Rapid Pathogen Detectioncai_hong@lanl.gov
(O): 505-606-1633 (Powerpoint
Presentation), Systems Integration in Biodefense Washington, D.C.Aug 22,
2006. For additional information, contact: Hong Cai: (E):
There is a significant need to expand nucleic acid sequence-based
amplification (NASBA) technologies ( NASBA® compared to RT-PCR®).
NASBA is an isothermal nucleic acid amplification process involving
the simultaneous activity of three enzymes; reverse transcriptase, RNase
H and T7 RNA polymerase (Compton, 1991; Kievits et al., 1991), thus mimicking
the process of retroviral replication (Compton, 1991). The technique utilises
two oligonucleotide primers in which the downstream antisense primer contains
a highly conserved 5’ promotor sequence recognized by T7 RNA polymerase.
Since reverse transcriptase is incorporated into the amplification mixture,
RNA can be added directly to amplification reactions without prior manipulation
such as the generation of cDNA templates, as are required for RT-PCR, thus
providing a single-tube amplification format. With NASBA, contaminating
double stranded DNA, which is often a problem in RT-PCR assays, is not denatured
at the isothermal amplification temperature (41°C) and therefore does not
participate in the amplification procedure, obviating the need for rigorous
RNA purification (Deiman et al., 2002). In veterinary virology, NASBA has
been used for the detection of several RNA viruses (Romano et al., 1996; Lanciotti and Kerst, 2001; Collins et al., 2002;
Jordan et al., 2002). -- Diagnostic Techniques
and Vaccines for Foot-and-Mouth Disease, Classical Swine Fever, Avian
Influenza and some other important OIE List A Diseases, European Commission
Report of the Scientific Committee on
Animal Health and Animal Welfare, April 2003.
References:
1. Advances
in molecular diagnostics for avian influenza: Dev Biol (Basel). 2006;124:93-7.
2. Real-Time
NASBA Detection of SARS-Associated Coronavirus and Comparison With Real-Time
Reverse Transcription-PCR, Journal of Medical Virology 77:602–608
(2005).
3. Real-time
NASBA detection of SARS-associated coronavirus and comparison with real-time
reverse transcription-PCR: J Med Virol. 2005 Dec;77(4):602-8.
4. Real-Time
Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid
Detection of West Nile Virus: Journal of Clinical Microbiology, January
2004, p. 257-263, Vol. 42, No. 1.
5. Nucleic
acid sequence-based amplification methods to detect avian influenza virus:
Biochem Biophys Res Commun. 2004 Jan 9;313(2):336-42.
6. Development
and evaluation of a real-time nucleic acid sequence based amplification
assay for rapid detection of influenza A: J Med Virol. 2004 Dec;74(4):619-28.
7. Development
and comparison of the real-time amplification based methods--NASBA-Beacon,
RT-PCR taqman and RT-PCR hybridization probe assays--for the qualitative
detection of sars coronavirus: Southeast Asian J Trop Med Public Health.
2004 Sep;35(3):623-9.
8. A
rapid biosensor for viable B. anthracis spores: Anal Bioanal Chem.
2004 Sep;380(1):15-23. Epub 2004 Aug 7.
9. Rapid
and sensitive detection of avian influenza virus subtype H7 using NASBA:
Biochem Biophys Res Commun. 2003 Jan 10;300(2):507-15.
10. A
NASBA method to detect high- and low-pathogenicity H5 avian influenza
viruses: Avian Dis. 2003;47(3 Suppl):1069-74.
11. Comparison
of nucleic acid-based detection of avian influenza H5N1 with virus isolation:
Biochem Biophys Res Commun. 2003 Mar 7;302(2):377-83.
12. Detection
of highly pathogenic and low pathogenic avian influenza subtype H5 (Eurasian
lineage) using NASBA: J Virol Methods. 2002 May 16;103(2):213-25.
13. Nucleic
Acid Sequence-Based Amplification Assays for Rapid Detection of West
Nile and St. Louis Encephalitis Viruses: Journal of Clinical Microbiology,
December 2001, p. 4506-4513, Vol. 39, No. 12.
I will be developing a molecular diagnostics technologies resource
to assist UN member countries engaged in medical/veterinary infrastructure
optimization. Companies that would like to collaborate in this initiative
can contact me via email: s.m.apatow@pathobiologics.org.
or phone: 203-668-0282 .
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