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A microwell-plate-based PCR assay is sensitive and specific for the detection of M. genitalium.

Development and performance of a microwell-plate-based polymerase chain reaction assay for Mycoplasma genitalium.
Dutro SM, Hebb JK, Garin CA, Hughes JP, Kenny GE, Totten PA.
Sexually Transmitted Diseases 2003;30:756-763.
 
Summary:

Question
How well does a microwell-plate-based PCR assay using biotinylated primers that target the MgPa gene and a dioxigenin-labeled probe, compare to a Southern-blot-based PCR assay using the same primers without biotin and a biotinylated probe, for the detection of M. genitalium?

Design
This study describes the development and evaluation of a microwell-plate-based PCR assay that uses colorimetric detection of amplicons, incorporates an internal inhibition control, and was adapted from a previously described M. genitalium MgPa gene Southern-blot-based PCR assay. The performance of the new assay was compared to that of the original assay for the detection of M genitalium in cervical specimens and in male urine specimens that were processed by different methods.

Participants
Specimens from two clinical studies, which had been previously analyzed using the original MgPa Southern-blot-based (SB) PCR assay, were used to evaluate the new assay: 1) 100 cervical exudate specimens (50 SB PCR positive and 50 negative) from women seen at the Seattle STD clinic, and 2) 64 male urine specimens (32 SB PCR positive and 32 negative). Cervical specimens were originally processed using the AMPLICOR CT/NG specimen preparation kit according to the manufacturer's directions (Roche Diagnostics Systems, Branchburg NJ, swab procedure). If the specimen was inhibited in the original PCR assay, it was diluted 1:5 and reanalyzed or further purified by the MasterPure DNA Purification kit according to the manufacturer's instructions (Epicentre, Madison, WI). The purified, uninhibited cervical specimens were used to set cutoff levels of the new assay and to evaluate its ability to detect M. genitalium.

Description of Tests and Diagnostic Standard
The same MgPa gene primer and probe sequences were used for both the new microwell-plate-based PCR assay (MW) and the original Southern-blot-based PCR assay (SB). The MW assay primers were labeled with biotin and amplification was performed in a 96-well thermocycler. Biotinylated PCR products were bound to streptavidin-coated microwell plates and detected with a digoxigenin labeled probe, enzyme-labeled antibody, and colorimetric substrate using the PCR ELISA kit (Roche). Color production was quantified at 405 nm. For the SB assay, unlabeled primers were used and amplification was performed in a 48-well format. MgPa PCR amplicons (258 bp) were detected by Southern blot hybridization using a biotinylated probe. Both assays included in the MgPa PCR reaction mixture 32 copies of an internal control plasmid, which contained the MgPa PCR product with the Mg probe binding portion replaced with a larger piece of DNA from the hemolysin gene of H. ducreyi. The internal control amplicon (630 bp) was amplified by the MgPa primers and detected using a specific digoxigenin-labeled probe in a separate well of the microwell plate for the MW assay and by hybridization with a specific biotin labeled probe after detection of the MgPa amplicon in the SB assay.

To determine the limit of detection of the MW assays, 16 replicate reactions each of serial 2-fold dilutions containing 128 to 1 copy of M. genitalium DNA and 64, 32 or 16 copies of internal control plasmid were analyzed using a positive cut-off of 0.10 OD.

Three specimen processing procedures were used to prepare the urine specimens for PCR analyses. 1) Urine samples were centrifuged, and the pellets were suspended in buffer and frozen. Thawed specimens were centrifuged again and the pellets suspended in lysis buffer, proteinase K, and Instagene matrix (Bio-Rad Laboratories, Hercules, CA). 2) Urine specimens were processed according to the AMPLICOR CT/NG specimen preparation kit (Roche, urine procedure). 3) Urine was processed with the MasterPure DNA Purification kit (Epicentre) according to the manufacturer's instructions. Urine was processed by the Instagene method on the day of collection and tested by the SB assay. Frozen urine specimens were processed by the Roche and Epicentre methods and analyzed by the MW assay. The amount of original urine specimen added to the PCR assays was 80 μl, 50 μl, or 16 μl for the Instagene matrix, Roche, and Epicentre specimen preparation methods, respectively.

Main Outcome Measures
The limit of detection of M. genitalium DNA in the MW assay and the agreement between the MW and SB assays were determined.

Main Results
The 95% detection limit for M. genitalium DNA was 13.4 and 15.5 copies for the MW assay without and with the internal control, respectively. The limit of detection in the assay without the internal control was not significantly different from that of the assay that included the internal control.

The MW assay OD values for 45 of the 50 cervical specimens that were negative by the SB assay ranged from 0.00 to 0.10, while values for 5 were >0.25. The MW assay OD values for 47 of the 50 cervical specimens that were positive by the SB assay were >0.25. Three of the SB positive specimens had MW assay OD values between 0.10 and 0.25. The negative cut-off value was set at <0.10, the positive cut-off value at >0.25, and the equivocal zone was set at >0.10 and <0.25. All positive specimens were repeat tested. All samples in the equivocal zone were repeated in duplicate; samples were positive if they had 1 positive or 2 equivocal values on repeat analyses. The 5 SB negative samples that were MW positive were negative when repeated and thus classified as negative. The 3 SB positive samples that were MW equivocal were classified as positive after being repeated in duplicate. Therefore, all 50 SB positive and all 50 SB negative cervical specimens were concordant by the MW assay. The cut-off value for the internal control was set at <0.25 for inhibited specimens. All of the 100 cervical specimens had internal control ODs >0.25.

Of the 32 urine specimens prepared by the Instagene matrix method and positive by the SB assay, 25 (78%) specimens prepared by the Roche method and 24 (75%) prepared by the Epicentre method were positive by the MW PCR after resolving equivocal specimens by repeat analyses. All 32 SB negative urine specimens were also negative by the MW assay irrespective of the specimen processing method. Five specimens processed by the Roche method were initially inhibited for amplification. They were M. genitalium negative and no longer inhibited after freezing, thawing and retesting. The MW assay was 89% (57/64) and 88% (56/64) concordant (kappa = 0.78 and 0.75) with the SB assay for urine specimens processed by the Roche and Epicentre methods, respectively. Nineteen urine specimens processed by the Epicentre method were tested by the SB PCR. One of 9 initially SB positive/MW negative, 5 of 5 concordant positive, and none of 5 concordant negative specimens were positive by the repeat SB assay.

Authors' Conclusions
The M. genitalium microwell-plate-based PCR assay is a high-throughput assay that is adapted to a 96-well format and incorporates an internal control. The advantages of this assay compared to the Southern-blot-based PCR assay are shorter turnaround time (2 days vs. 4 days), less subjective positive and negative cutoff values, and the detection of samples containing inhibitors of amplification. All positive specimens should be repeated to control for contamination or sampling errors.

Source of funding: Grants from the National Institute of Allergy and Infectious Diseases

For correspondence: Patricia A. Totten, Department of Medicine, Harborview Medical Center, Box 359779, 325 9th Ave., Seattle, WA 98104. E-mail address: patotten@u.washington.edu.

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