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Milford Medical
Laboratory provides comprehensive testing capabilities and personal
service for tens of thousands of patients at three Patient Service
Centers in Milford and Orange. The laboratory is an affiliate of
Milford Hospital. Milford Medical Laboratory has modern
instrumentation and advanced equipment run by trained and experienced
specialists. With early hours, three convenient locations, and no
appointment necessary, the laboratory is designed for convenience.
Locations and Hours:
30 Commerce Park Rd., Milford
Open: 8 a.m. to Noon, and 1-3 p.m.
(203) 876-7440
339 Boston Post Rd., Orange (corner of Lambert Road)
Open: 8 a.m. to Noon, and 1-5 p.m.
(203) 799-0301
2068 Bridgeport Ave., Milford (opposite Milford Hospital)
Open: 7 a.m. to 5 p.m.
(203) 876-7745
Milford Medical Laboratory (MML) of
Milford, CT is certified to perform high complexity tests by the
Connecticut Department of Health and the Centers for Medicare &
Medicaid Services under the Clinical Laboratory Improvement Act of
1988. Its molecular diagnostics department has introduced the nation’s
first routine DNA sequencing-based no-false positive HPV genotyping,
Neisseria gonorrhoeae opa gene DNA amplification assay, and Chlamydia
trachomatis cryptic plasmid DNA amplification assay for women’s health
care management. The nucleic acids of these three sexually transmitted
infectious agents can be detected in a single liquid-based Pap cytology
specimen (Cytyc or Surepath) with targeted nested PCR amplification,
followed by direct DNA sequencing for final validation.
MML is a leader in developing molecular
diagnostic procedures to facilitate transferring the advanced
PCR/direct DNA sequencing biotechnology into community hospital
laboratories. The following services are provided:
- Performance of HPV genotyping, N.
gonorrhoeae assay and C. trachomatis assay based on DNA sequencing, a
direct service to ordering physicians. MML will process the insurance
claims on behalf of the patients for the test services
- MML will perform the HPV genotyping, N.
gonorrhoeae assay and C. trachomatis assay based on DNA sequencing on
behalf of the referring hospitals or laboratories which will reimburse
the MML for the testing services.
- MML will assist CLIA-certified high
complexity laboratories in establishing in-house molecular programs
(after January 1, 2008) to perform the above tests, including:
- Economical feasibility studies
- Site assessment & design
- Equipment procurement and installation
- Establishment of test procedures
- Billing and reimbursement training
- Technical training
- Material ordering
- Assay validation consulting
- Preparation for inspections
- Continued technical support
For more information, contact
George T. Poole, MT(ASCP), MS, MPA
203-876-4496
george.poole@milfordhospital.org
SECTION II
New technical procedures developed by the Molecular Diagnostics Department of MML:
HPV L1 gene DNA amplification
(1) Primary PCR amplification of HPV L1 gene DNA
Into a 0.2 mL PCR tube, pipette 1µL of
positive HPV-16 DNA control or a cell digestate containing HPV DNA.
Then add 24 µL of enzyme/primer reaction mixture consisting of 20µL
HiFi® LoTemp™ PCR ready-to-use mix, 2 µL water, 1µL HPV outer PCR
forward primer and 1µL HPV outer PCR reverse primer. The standard
LoTemp™ PCR thermocycling is programmed as below.
Initial 85ºC 1 cycle 10 min.
85ºC 30
sec
40ºC 30
sec
65ºC
1 min for 30 cycles
Final extension 65ºC for 10 minutes.
(2) Nested PCR of HPV L1 gene DNA
Into a 0.2 mL PCR tube, pipette 25µL of
the complete enzyme/primer nested PCR mixture consisting of 20µL HiFi®
LoTemp™ PCR ready-to-use mix, 3 µL water, 1µL HPV nested PCR forward
primer and 1µL HPV nested PCR reverse primer. Transfer a trace of the
primary PCR product from the primary PCR tube with a glass rod of about
1.1-1.5 mm in diameter into the complete enzyme/primer nested PCR tube.
Run the standard LoTemp™ cycling.
(3) Gel electrophoresis of PCR products
After performing agarose gel
electrophoresis, visualization of an ~190 bp amplicon on the nested PCR
lane with or without a 450 bp amplicon in the corresponding primary PCR
lane of the sample provides presumptive evidence that the nested PCR
system has successfully amplified a segment of HPV L1 gene DNA which is
present in the sample. Final validation of the nested PCR products is
accomplished by direct DNA sequencing with a standard automated
fluorescent dye-labeled terminator reaction, using an HPV DNA
sequencing general primer. (see signature sequence )
Alternatively, the consensus MY09/MY11
primer and the GP5+/GP6+ general primary pairs can be use for the
primary and nested PCR respectively; and the GP6+ can be used as the
sequencing primer, as reported in http://www.infectagentscancer.com/content/2/1/11
Neisseria gonorrhoeae opa gene DNA amplification
(1) Primary PCR amplification of gonococcal opa gene DNA
Into a 0.2 mL PCR tube, pipette 1µL of
Neisseria gonorrhoeae DNA control or a cell digestate containing
Neisseria gonorrhoeae DNA. Then add 24 µL of enzyme/primer reaction
mixture consisting of 20µL HiFi® LoTemp™ PCR ready-to-use mix, 2 µL
water, 1µL N. gonorrhoeae opa gene outer PCR forward primer and 1µL N.
gonorrhoeae opa gene outer PCR reverse primer. Run the standard LoTemp™
thermocycling.
(2) Nested PCR of gonococcal opa gene DNA
Into a 0.2 mL PCR tube, pipette 25µL of
the complete enzyme/primer nested PCR mixture consisting of 20µL HiFi®
LoTemp™ PCR ready-to-use mix, 3 µL water, 1µL N. gonorrhoeae opa gene
nested PCR forward primer and 1µL N. gonorrhoeae opa gene nested PCR
reverse primer. Transfer a trace of the primary PCR product from the
primary PCR tube with a glass rod of about 1.1-1.5 mm in diameter into
a complete enzyme/primer nested PCR tube. Run the standard LoTemp™
cycling.
(3) Gel electrophoresis of PCR products
After performing agarose gel
electrophoresis, visualization of a 127-130 bp amplicon on the nested
PCR lane with or without a 210 to 270 bp amplicon(s) in the
corresponding primary PCR lane of the sample provides presumptive
evidence that the nested PCR system has successfully amplified a
promoter segment of the species-specific gonococcal opa genes which are
present in the sample. Final validation of the nested PCR products is
accomplished by direct DNA sequencing with a standard automated
fluorescent dye-labeled terminator reaction, using the gonococcal opa
gene sequencing primer. (see signature sequence )
Chlamydia trachomatis cryptic plasmid DNA amplification
(1) Primary PCR of Chlamydia trachomatis cryptic plasmid DNA
Into a 0.2 mL PCR tube, pipette 1µL of
Chlamydia trachomatis DNA control or a cell digestate containing
Chlamydia trachomatis DNA. Then add 24 µL of enzyme/primer reaction
mixture consisting of 20µL HiFi® LoTemp™ PCR ready-to-use mix, 2 µL
water, 1µL C. trachomatis plasmid outer PCR forward primer and 1µL C.
trachomatis plasmid outer PCR reverse primer. Run the standard LoTemp™
thermocycling.
(2) Nested PCR of Chlamydia trachomatis cryptic plasmid DNA
Into a 0.2 mL PCR tube, pipette 25µL of
the complete enzyme/primer nested PCR mixture consisting of 20µL HiFi®
LoTemp™ PCR ready-to-use mix, 3 µL water, 1µL C. trachomatis cryptic
plasmid nested PCR forward primer and 1µL C. trachomatis cryptic
plasmid nested PCR reverse primer. Transfer a trace of the primary PCR
product from the primary PCR tube with a glass rod of about 1.1-1.5 mm
in diameter into a complete enzyme/primer nested PCR tube. Run the
standard LoTemp™ cycling.
(3) Gel electrophoresis of PCR products
After performing agarose gel
electrophoresis, visualization of an ~160 bp amplicon on the nested PCR
lane with or without an ~290 bp amplicon in the corresponding primary
PCR lane of the sample provides presumptive evidence that the nested
PCR system has successfully amplified a segment of C. trachomatis
plasmid DNA which is present in the sample. Final validation of the
nested PCR products is accomplished by direct DNA sequencing with a
standard automated fluorescent dye-labeled terminator reaction, using
the Chlamydia trachomatis cryptic plasmid DNA sequencing primer. (see signature sequence )
Automated DNA sequencing was performed
with BigDye® Terminator (v 1.1/Sequencing Standard Kit) according to
the protocol supplied by Applied Biosystems Inc. See more information
related to press release of July 23, 2007.
SECTION III
DNA sequencing tests for human papillomavirus(HPV),
Chlamydia trachomatis and Neisseria gonorrhoeae
As of July 2007, the Molecular
Diagnostic Department of MML offers “no false positive” DNA sequencing
tests for the three common sexually transmitted infections (STIs) to
gynecologists and their patients. All three tests, for anyone or in any
combination of the three, including HPV genotyping
, C. trachomatis and N. gonorrhoeae can be performed on one
liquid-based Pap cytology specimen (Cytyc or Surepath) by order of a
referring physician. The DNA of these causative agents is individually
amplified by LoTemp™ polymerase chain reaction (PCR) for detection and
the positive result is validated by DNA sequencing. The signature sequence specific for each identified agent is enclosed in the final report to the physician for clinical follow-ups.
DNA sequencing eliminates the false
positive results associated with other DNA testing methods. A false
positive test result of STIs can have adverse medical, social and
psychological impacts for a patient.
Physicians and patients interested in
this new test, or pick-up service if needed, may call (203) 876-4264 or
876-7745. Physicians may also download the requisition form by clicking here
and then send in with the specimen. Milford Hospital or MML will
process the insurance claims on behalf of the patients for the test
services.
Milford Medical Laboratory Offers No-False-Positive DNA-sequencing Human Papillomavirus (HPV), Chlamydia and Gonococcus Tests
All-in-1 liquid-based Pap cytology specimen tested with a new HiFi® LoTemp™ PCR Technology
MILFORD, Conn. -- The nation’s
first routine test for sexually transmitted infections (STIs) using DNA
sequencing which eliminates the false positive test results associated
with other methods, is now available to physicians and their patients.
A false positive diagnosis of STIs can
have adverse medical, social and psychological impacts on a patient,
said Sin Hang Lee, MD, who initiated and developed the DNA sequencing
test based on a new LoTemp™ PCR amplification technology. With DNA
sequencing, once a signature sequence is identified, it can only come
from one specific organism, thus eliminating all false identifications.
DNA sequence, confirmed by the national GenBank database, is like the
“Social Security Number” of a microbe.
HPV, chlamydia and gonococcus are the
causative agents for the three most common STIs in the United States.
Early accurate diagnosis of STIs is important for proper health care in
women. As of July 2007, the U.S. Preventive Services Task Force
(USPSTF) recommends screening for chlamydial infection for all sexually
active or pregnant women aged 24 and younger, and for older pregnant
women who are at increased risk, so that timely treatment can be
instituted. The USPSTF also cautions the physicians that in low
prevalence populations a positive test is more likely to be a false
positive than a true positive. This is because the DNA test kits
currently on the market were originally developed for screening
populations with high prevalence of STIs, all with a few percentages
margin of error. In low prevalence populations, the number of false
positive results generated by these test kits exceeds that of the true
positives. In contrast, a DNA sequencing assay is self-validated,
automatically eliminating any false positive results.
The first scientific report on routine HPV genotyping by DNA sequencing was just published in Infectious Agents and Cancer.
The second manuscript on testing all three STIs is under peer review.
Accurate HPV testing is important in following persistent HPV
infections which may lead to cervical cancer. For example, HPV-66, a
known carcinogenic genotype, is relatively common in New Haven County,
but not targeted for detection by the commonly used Digene HC2 assay.
HPV genotyping information is also useful in monitoring HPV infections
before and after vaccine immunization to develop prevention strategy
for the individual patients. Dr. Lee has petitioned the Food and Drug Administration
to down-classify the HPV tests to class II virology device category to
facilitate competitive introduction by small innovative manufacturers
of their new technologies via the 510K applications in compliance with
the least burdensome provisions of the FDA Modernization Act of 1997.
The molecular diagnostic procedures
performed at Milford Medical Laboratory are approved as high complexity
tests by the State of Connecticut Department of Health and by the
Centers for Medicare & Medicaid Services under the Clinical
Laboratory Improvement Act of 1988, said Dr. Pappu.
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