|Year : 2013 | Volume
| Issue : 1 | Page : 15-18
Alteration in sample preparation to increase the yield of multiplex Polymerase Chain Reaction assay for diagnosis of genital ulcer disease
G Rao1, A Das2, P Prabhakar2, V Nema1, AR Risbud1
1 Department of Microbiology, National AIDS Research Institute, 73 'G', MIDC, Bhosari, Pune-411 026, India
2 Department of Microbiology, Family Health International, 360, New Delhi, India
|Date of Submission||26-May-2012|
|Date of Acceptance||24-Oct-2012|
|Date of Web Publication||15-Mar-2013|
A R Risbud
Department of Microbiology, National AIDS Research Institute, 73 'G', MIDC, Bhosari, Pune-411 026
Source of Support: Financial Assistance from Bill and Melinda Gates Foundation for STI- OR study among HRGs., Conflict of Interest: None
Purpose: Genital Ulcer Disease (GUD) is common sexually transmitted infection (STI). Multiple studies have shown that GUDs are strongly associated with the transmission and the acquisition of HIV infection. An accurate diagnosis of common etiology of GUD namely Herpes, syphilis and Chancroid is possible using Multiplex PCR (M-PCR). However, frequent presence of Polymerase Chain Reaction inhibitors in the ulcer swab specimen limits the performance of the assay. In order to overcome this problem, alternative specimen preparation method was used. Materials and Methods: To determine the common etiology, GUD specimens obtained under an STI operations research study were tested with M-PCR after the samples were prepared using Roche Amplicor specimen preparation kit. PCR inhibiting samples were identified from that, which showed negative results. These samples were subjected to phenol-chloroform extraction and ethanol precipitation before the conduct of M-PCR on them. Results: Of the 237 GUD specimens tested, in 145 etiologies could be detected, whereas 92 samples were found negative. Further spiking with one of the target DNA, 128 of the negative samples were found to contain the inhibitors. These 126 samples were then subjected to phenol chloroform extraction and ethanol precipitation followed by M-PCR. Using this method for sample preparation, etiology could be determined in 46 (23%) additional samples. This success rate of altered sample preparation method has been lower than that has reported. Conclusion: The results indicate that sample preparation using phenol chloroform extraction and ethanol precipitation, prior to M-PCR helps to eliminate the inhibitors and increase the yield of the assay. However, being a laborious procedure, it may be used for samples giving negative results after the screening by Roche Amplicor specimen preparation kit.
Keywords: Genital ulcer disease, multiplex PCR, sample preparation
|How to cite this article:|
Rao G, Das A, Prabhakar P, Nema V, Risbud A R. Alteration in sample preparation to increase the yield of multiplex Polymerase Chain Reaction assay for diagnosis of genital ulcer disease. Indian J Med Microbiol 2013;31:15-8
|How to cite this URL:|
Rao G, Das A, Prabhakar P, Nema V, Risbud A R. Alteration in sample preparation to increase the yield of multiplex Polymerase Chain Reaction assay for diagnosis of genital ulcer disease. Indian J Med Microbiol [serial online] 2013 [cited 2019 Mar 26];31:15-8. Available from: http://www.ijmm.org/text.asp?2013/31/1/15/108709
| ~ Introduction|| |
Genital Ulcer diseases (GUDs) are the common sexually transmitted infections (STIs). , Multiple studies have shown strong association of GUDs with transmission and acquisition of HIV infection.  Treponema pallidum (T. pallidum), Haemophilus ducreyi (H. ducreyi) and Herpes simplex virus (HSV) infection are the common etiologies of GUDs, of which, the first two are completely curable.
PCR and especially, multiplex PCR (M-PCR) assay has been shown to be a reliable alternative to conventional methods.  However, M-PCR may have limitations in multiple and mixed infections for detection of specific GUD etiologies.  Additionally, the PCR may fail to detect the etiology in about 20-30% of the specimens, which may be due to presence of PCR inhibitors in the specimens. , Sample preparation method used prior to PCR may help to overcome this problem.
The present study aimed at assessing the altered methodology of specimen preparation in increasing the performance of M-PCR assay for detection of GUD etiology among Female Sex Workers (FSW), Men having Sex with Men (MSM) and Male STI patients in India.
| ~ Materials and Methods|| |
The present study formed a part of a larger study to evaluate the sexually transmitted infection service package for FSWs, MSMs and Male STI patients in India. In order to determine the etiology of GUD syndrome among these populations, a total of 237 (FSWs 18, MSMs 9 and Male STI patients 210) individuals, presenting with genital ulcers, were recruited from seven dedicated clinics for FSW and MSM in two states (Andhra Pradesh, Maharashtra) and 8 STD clinics from states of Maharashtra, Andhra Pradesh, Delhi and Manipur from India.
This study was approved by the Ethics committee of the National AIDS Research Institute and the Protection of Human Subjects Committee of FHI, North Carolina, USA.
Behavioral and clinical data was collected by administering a structured questionnaire (data not shown).
Specimen collection and sample processing
GUD ulcers were cleaned with sterile Dacron swab (Fischer Scientific, USA) moistened with saline and ulcer was scraped and swab was collected in 2SP transport medium and stored at study site in -20 o C. In case of more than one ulcer, a separate swab was collected per ulcer. Samples were then transported on dry ice to the National AIDS Research Institute, Pune, India, where they were frozen and stored at -70 o C until tested.
GUD swabs were processed by Roche Amplicor specimen preparation kit (Roche Molecular Diagnostics, California, USA).  In brief, micro centrifuge tubes were labeled for each swab sample. Hundred micro liter CT/NG Lysis buffer was added to each of the empty labeled tubes. Hundred micro liters well mixed GUD specimens were added to micro centrifuge tubes containing 100 μl CT/NG Lysis buffer by using new aerosol barrier tip for each specimen. All tubes were recapped immediately and mixed well by vortexing and kept for incubation at room temperature for 15 minutes. Using a new aerosol barrier pipette tip for each specimen, 200 μL CT/NG diluent was added to each tube and incubated at room temperature for 15 minutes. M-PCR was performed on these prepared samples for detection of T. pallidum, H. ducreyi and HSV etiology as described below. 
Polymerase Chain Reaction amplification
PCR amplification was performed with Applied Biosystems PCR System 9600. The M-PCR mixture contained 50 μl of processed swab specimen and 50 μl of master mixture that resulted in the following final concentrations of reagents: 25 pmol each of the three pairs of biotinylated oligonucleotide primers [Table 1] (Bioresource, Bangalore, India). 10X Tris buffer, 1.5 mM MgCl 2 (already present in specimen diluent), 200 mM (each) dTTP, dATP, dGTP, and dCTP, 3U of Taq polymerase (Bangalore Genei, Bangalore, India). PCR cycling conditions followed were: 92 o C for 5 min, 92 o C for 20 sec, 62 o C for 20 sec, 72 o C for 20 sec, 72 o C less than 2 hours and 35 cycles.
Detection of PCR products
Three micro well strips (Costar, Lowell, USA) were conjugated via 5' end of oligonucleotide capture probes specific for each target to the bottom of separate micro wells using freshly prepared 1M ammonium acetate.  Detection and identification of all three targets were achieved by colorimetric detection system with AMPLICOR detection kit. Specimens were scored as positive for a specific pathogen if duplicate reactions produced signals with OD of ≥0.8 at 450 nm. Specimens with split positive and negative results were re-amplified in duplicate and were reanalyzed until the replicates were concordant. Specimens giving negative results for all three targets were processed for detection of presence of PCR inhibitors.
Identification of PCR Inhibitors in the negative specimens
All target negative samples were spiked with 100 copies of one of the positive controls (H. ducreyi -DNA) and were tested by M-PCR as described above. Specimens showing OD of ≥0.8 at 450 nm for H. ducreyi DNA were considered free of PCR inhibitors. Whereas, specimen showing negative signal (at 450 nm signal <0.25) were considered to have PCR inhibitors. All specimen identified as inhibitory were processed by altered sample preparation method of phenol chloroform extraction and ethanol precipitation  followed by M-PCR.
Phenol-Chloroform extraction and ethanol precipitation
GUD specimen were thawed and brought to room temperature. By using plugged, elongated pipette tip, specimens (100 μl each) were added to a screw cap tube having 400 μl of AMPLICOR Specimen Transport Medium which contained 1 μg/ml Thymus calf DNA. The tubes were vortexed and heated at 95°C for 10 min, then cooled to room temperature and pulse-centrifuged. Five hundred micro liters phenol:chloroform:isoamyl alcohol (25:24:1) (SD fine, Mumbai, India) was added, mixed and centrifuged for 10 min at 15000 rpm. Upper aqueous layer was transferred to a fresh micro centrifuge tube containing 500 μl chloroform: isoamyl alcohol (24:1), mixed and centrifuged for 10 min at 15000 rpm. Aqueous layer was transferred to fresh tube containing 500 μl of 3 M sodium acetate, pH 5.2. This was mixed and 1000 μl of cold 100% ethanol was added to it. This mixture was incubated overnight at -20°C for DNA precipitation. After overnight incubation, samples were centrifuged for 20 min at 15000 rpm at 4°C.
Supernatant was removed completely and pellet was dried by incubating the open tube in laminar airflow. Pellet was re-suspended in 120 μl of a 1:1 mixture of AMPLICOR specimen transport medium and AMPLICOR specimen diluent.
| ~ Results|| |
Of the 237 swabs, processed by Roche Amplicor specimen preparation kit followed by M-PCR assay, GUD etiology was determined in 91 (38%) cases. Among these, 22 T. Pallidum, 60 HSV and 9 mixed infections (T. Pallidum and HSV) were detected. Of the 146 samples, 6 specimens gave equivocal results and were not considered for further testing. Among the remaining 140 samples, negative for all three targets, 12 specimens were found to be true negative by showing positive signal with the spiked H. ducreyi DNA. Finally, 128 (54%) GUD samples were considered to have PCR inhibitors since no positive signal was observed in spite of spiking with H. ducreyi DNA. Among these, volume of two specimens was insufficient and hence 126 specimens were further processed by phenol chloroform extraction method and M-PCR was done in duplicate. Using this method, etiology could be determined in 46 additional cases (T. Pallidum 16, HSV 29, H. ducreyi 1 and mixed infections 8 - T. Pallidum and HSV). Thus, after using altered sample preparation method, etiology of GUD could be determined in a total of 145 (61%) specimen. Of the 3 common etiologies, HSV was found in 37.5 % followed by T. Pallidum in 16% and H. ducreyi in 0.42%. Dual infection of T. Pallidum and HSV were seen in 17 (7%) cases [Table 2].
|Table 2: Multiplex PCR results before and after phenol chloroform extraction|
Click here to view
| ~ Discussion|| |
Sexually transmitted infections constitute a major health problem in developed and developing countries. In particular, GUD syndrome has been associated with increased risk for HIV infection and transmission.  Since two of these 3 common infections viz. T. Pallidum, H. ducreyi can be completely cured by treatment, accurate and early diagnosis is important for effective control of these infections. This can be achieved by most sensitive and specific diagnostic technique like polymerase chain reaction.  A multiplex PCR (M-PCR) for simultaneous detection of H. ducreyi, T. Pallidum and HSV has been proved to be highly sensitive when compared to other standard diagnostic tests. However, M-PCR fails to detect etiology in about 20-30% specimen.
Specimen when tested, using Roche Amplicor sample preparation kit followed by M-PCR, etiology could be determined only in 38% of cases. Since large number of samples gave negative results, they were tested for the presence of inhibitors. By spiking all target negative specimen (140) with H. ducreyi DNA, we observed that 128 (54%) of them were suspected to have PCR inhibitors. This proportion of samples having PCR inhibitors was much higher as compared to reports from other studies, where, around 11 to 20% of the samples were undiagnosed due to PCR inhibitors. , Though, the precise reason for such a large number of samples with inhibitors could not be ascertained, it may have been due to the topical self-application of some medication. The other reason could be a long storage of swabs in the transport medium for about 12 months at-70°C before testing. Similarly, of the 126 samples found to have inhibitors, only in 23% of them etiology could be detected even after phenol-chloroform extraction. This success rate also is lesser to what has been reported in other studies. , A large number of negative samples with PCR inhibitors, observed in the study, could be one of the reasons for this low success.
In the present study, GUD specimen giving negative results in the M-PCR assay, were re- extracted by phenol chloroform and were precipitated using ethanol. This pre-treatment of the specimen enhanced the performance of the M-PCR assay and resulted in detection of etiology in 46 (23%) additional GUD samples.
Overall, in 38% samples, the etiology could not be determined. It may be due to other reasons than those mentioned above, which include, presence of etiology other than those investigated, inappropriate or inadequate sample collection, limitations of the sample preparation method used etc.
| ~ Conclusion|| |
The results of the study indicate that Roche Amplicor sample preparation, being a less cumbersome procedure, could initially be used to screen large number of samples and all target negative specimen, be further processed by phenol chloroform extraction and ethanol precipitation before undertaking M-PCR to increase the yield.
| ~ Acknowledgments|| |
This study was funded by The Bill and Melinda Gates Foundation. The views expressed herein are those of the authors and do not necessarily reflect the official policy or position of The Bill and Melinda Gates Foundation.
We thank Dr. Ian Maclean University of Manitoba, Canada for providing probe and probe controls of target organisms.
| ~ References|| |
|1.||Gangakhedkar R, Risbud AR, Gadkari DA, Chan-Tack K, Shepherd ME, Bollinger RC, et al. Genital ulcer disease etiology by M-PCR in STD patients in Pune, India: Implications for syndromic management. International Conference on AIDS. Int Conf AID 1998;12:302. |
|2.||Risbud A, Chan-Tack K, Gadkari D, Gangakhedkar RR, Shepherd ME, Bollinger R, et al. The etiology of genital ulcer disease by multiplex polymerase chain reaction and relationship to HIV infection among patients attending sexually transmitted disease clinics in Pune, India. Sex Transm Dis 1999;26:55-62. |
|3.||Dickerson MC, Johnstone J, Delea TE, White A, Andrews E. The causal role for genital ulcer disease as a risk factor for transmission of human immunodeficiency virus. Sex Transm Dis 1996;23:429-40. |
|4.||Orle KA, Gates CA, Martin DH, Body BA, Weiss JB. Simultaneous PCR detection of Haemophdus ducreyi, Treponema pallidum, and Herpes Simpex virus types 1 and 2 from geital ulcers. J Clin Microb 1996;34:49-54. |
|5.||Morse SA, Trees DL, Htun Y, Radebe F, Orle KA, Dangor Y, et al. Comparison of clinical diagnosis and standard laboratory and molecular methods for the diagnosis of genital ulcer disease in Lesotho: Association with human immunodeficiency virus infection. J Infect Dis 1997;175:583-9. |
|6.||Clewly JP. The polymerase chain reaction: A review of the practical limitations of human immunodeficiency virus diagnosis. J Virol Methods 1989;25:179-88. |
|7.||Kolk AH, Schuitema AR, Kuijper S, van Leeuwen J, Hermans PW, van Embden JD, et al. Detection of Mycobacterium tuberculosis in clinical samples by using the polymerase chain reaction and a nonradioactive detection system. J Clin Microbiol 1992;30:2567-75. |
|8.||Roche Molecular Diagnostics Product kit Insert. Available from: http://www.fda.gov/downloads/BiologicsBloodVaccines/SafetyAvailability/TissueSafety/ucm100246.pdf [Last accessed on 2010 Sep 25]. |
|9.||Karina A, Orle, Judith B. Weiss. Detection of Treponema palliduim, Haemophilus ducreyi, and Herpes Simplex Virus by Multiplex PCR, From Methods/n Molecular Methods. In: Peehng RW, Sparhng PF, editors. Sexually Transmitted Diseases Methods and Protocols. Vol. 20. Totowa, NJ: Humana Press Inc.; 1999. p. 67-79. |
|10.||Tung CH, Rudolf MJ, Stein S. Preparation of oligonucleotide- peptide conjugates. Bioconjugate Chem 1991;2:464-5. |
|11.||Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: A laboratory manual. 2 nd ed., vol. 3. New York: Cold Spring Harbor Laboratory Press; 1982. p. 89-96. |
|12.||Behets FM, Brathwaite AR, Hylton Kong T, Chen CY, Hoffman I, Wiss JB, et al. Genital ulcers: Etiology, clinical diagnosis, and associated human immunodeficiency virus infection in Kingston, Jamaica. Clin Infect Dis 1999;28:1086-90. |
|13.||Sweet RL, Gibbs RS. Infectious Diseases of the Female Genital Tract, 4 th ed. Philadelphia: Lippincott, Williams and Wilkins Press; 2002. p. 101-16. |
|14.||Jain A, Tiwari V, Guleria RS, Verma RK. Qualitative evaluation of mycobacterial DNA extraction protocols for polymerase chain reaction. Mol Biol Today 2002;3:43-50. |
[Table 1], [Table 2]