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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 85şC 1 cycle 10 min.
85şC 30 sec
40şC 30 sec
65şC 1 min for 30 cycles
Final extension 65şC 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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