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Sains Malaysiana 47(3)(2018): 531–536
http://dx.doi.org/10.17576/jsm-2018-4703-13
Saliva Sampling of Alcoholic Participants
using Three Saliva Collection Methods
(Persampelan Air Liur menggunakan Tiga Kaedah
Pengumpulan Air Liur daripada Peserta yang Mengamalkan Minuman Beralkohol)
KHAN, S.S., JAMEEL, R.A., RAZAK, F.A.
& BAKRI, M.M.*
University of Malaya, 50603
Kuala Lumpur, Federal Territory, Malaysia
Diserahkan: 6 April
2017/Diterima: 29 September 2017
ABSTRACT
The potential
of using saliva as a diagnostic fluid is well documented. The
aim of this study was to assess the quality and quantity of saliva
DNA of
alcoholic and non-alcoholic participants using three saliva collection
methods; DNA-SalTM
(Oasis Diagnostics, USA), Oragene-DNA (DNA Genotek
Inc, Ontario, Canada) and whole saliva collection method. Saliva
DNA
of non-alcoholic (n=30) and alcoholic participants (n=10) age between 25
and 35 years was assessed qualitatively and quantitatively using
spectrophotometry. Saliva DNA quantity was the highest for all participants when using
the DNA-Sal TM saliva collection kit (p<0.05).
The use of a mechanical scraper provided only in the DNA-Sal
TM
kit may have contributed to the highest DNA yield
for all participants. The quantity of saliva DNA when
assessed using spectrophotometer was found to be significantly
lower (p<0.05) for the alcoholic (16±3.57
ng/μL) than non-alcoholic participants (19.92±6.18
ng/μL). To determine the integrity of the DNA
samples, PCR
amplification of the Alcohol Dehydrogenase
gene, ADH1B was carried
out and the PCR was found to be successful. For all
participants, the DNA quality of the saliva collected using
the three saliva collection methods was found to be in the acceptable
range considered as pure DNA. The DNA quality
and quantity of saliva collected from the three saliva collection
methods were considered suitable for research purposes. Keywords: Alcohol;
PCR; saliva; saliva collection methods
ABSTRAK
Potensi menggunakan
air liur sebagai alat diagnostik telah pun mendapat pendedahan
yang meluas. Tujuan kajian ini adalah untuk mengkaji kualiti dan
kuantiti DNA
menggunakan sampel air liur yang diperoleh daripada
peserta yang mengamalkan minuman beralkohol. Sampel air liur diperoleh
daripada semua peserta menggunakan 3 kaedah pengumpulan air liur;
DNA-SalTM (Oasis
Diagnostics, USA), Oragene-DNA (DNA Genotek
Inc, Ontario, Canada) dan pengumpulan air liur secara langsung
daripada kaviti mulut. Persampelan air liur melibatkan peserta
yang tidak mengamalkan minuman beralkohol (n=30) dan yang mengamalkan minuman beralkohol (n=10) serta
berumur antara 25 dan 35 tahun. Kualiti dan kuantiti DNA air
liur daripada semua peserta dikaji menggunakan spektrofotometer.
Kuantiti DNA
air liur bagi semua peserta adalah paling tinggi
apabila menggunakan kaedah pengumpulan air liur DNA-Sal TM (p<0.05).
Penggunaan alat mengikis yang dibekalkan hanya untuk kaedah pengumpulan
air liur DNA-Sal TM didapati berkemungkinan menyumbang
terhadap kuantiti DNA yang paling tinggi. Walau
bagaimanapun, pemeriksaan spektrofotometer mendapati bahawa kuantiti
DNA
air liur bagi peserta yang mengamalkan alkohol (16±3.57
ng/μL) adalah lebih rendah (p<0.05) berbanding
dengan peserta yang tidak mengamalkan alkohol (19.92±6.18
ng/μL). Untuk memastikan integriti DNA
air liur, DNA yang diperoleh daripada air liur digunakan untuk amplifikasi
PCR. Amplifikasi PCR didapati telah berjaya bagi
salah satu gen kumpulan Alkohol Dehidrogenase, ADH1B.
Kualiti DNA air liur yang dikumpul menggunakan ketiga-tiga kaedah
pengumpulan air liur bagi semua jenis peserta didapati berada
dalam lingkungan yang dianggap sebagai DNA tulen. Kualiti dan kuantiti DNA
bagi air liur yang dikumpul menggunakan ketiga-tiga
kaedah pengumpulan air liur adalah dianggap sesuai bagi kegunaan
penyelidikan. Kata kunci: Air liur; alkohol;
kaedah pengumpulan air liur; PCR RUJUKAN
Abaz, J., Walsh, S.J.,
Curran, J.M., Moss, D.S., Cullen, J., Bright, J.A., Crowe, G.A., Cockerton,
S.L. & Power, T.E.B. 2002. Comparison of the variables affecting the recovery
of DNA from common drinking containers. Forensic Science International 126:
233-240.
Abraham, J.E., Maranian,
M.J., Spiteri, I., Russell, R., Ingle, S., Luccarini, C., Earl, H.M., Pharoah,
P.P., Dunning, A.M. & Caldas, C. 2012. Saliva samples are a viable
alternative to blood samples as a source of DNA for high throughput genotyping. BMC Medical Genomics 5: 19. doi:10.1186/1755-8794-5-19.
Ahn, S.M., Chan, J.Y.,
Zhang, Z., Wang, H., Khan, Z., Bishop, J.A., Westra, W., Koch, W.M. &
Califano, J.A. 2014. Saliva and plasma quantitative polymerase chain
reaction-based detection and surveillance of human papillomavirus-related head
and neck cancer. JAMA Otolaryngol Head Neck Surg. 140: 846-854.
doi:10.1001/jamaoto.2014.1338.
Birnboim, H.C. 2004. DNA
Yield with Oragene• DNA. DNA Genotek.
Chai, R.C., Lim, Y.,
Frazer, I.H., Wan, Y., Perry, C., Jones, L., Lambie, D. & Punyadeera, C.
2016. A pilot study to compare the detection of HPV-16 biomarkers in salivary
oral rinses with tumour p16(INK4a) expression in head and neck squamous cell
carcinoma patients. BMC Cancer 16: 178. doi:10.1186/s12885-016-2217-1.
Cozier, Y.C., Palmer,
J.R. & Rosenberg, L. 2004. Comparison of methods for collection of DNA
samples by mail in the black women’s health study. Annals of Epidemiology 14:
117-122.
Crabb, D.W., Edenberg,
H.J., Bosron, W.F. & Li, T.K. 1989. Genotypes for aldehyde dehydrogenase
deficiency and alcohol sensitivity. The inactive ALDH2(2) allele is dominant. The
Journal of Clinical Investigation 83: 314-316. doi:10.1172/jci113875.
Dawes, C. 1987.
Physiological factors affecting salivary flow rate, oral sugar clearance, and
the sensation of dry mouth in man. J. Dent. Res. 66: 648-653.
de Almeida Pdel, V.,
Gregio, A.M., Machado, M.A., de Lima, A.A. & Azevedo, L.R. 2008. Saliva
composition and functions: A comprehensive review. The Journal of
Contemporary Dental Practice 9: 72-80.
Fabryova, H. &
Celec, P. 2014. On the origin and diagnostic use of salivary RNA. Oral Dis. 20:
146-152. doi:10.1111/odi.12098.
Feigelson, H.S.,
Rodriguez, C., Robertson, A.S., Jacobs, E.J., Calle, E.E., Reid, Y.A., &
Thun, M.J. 2001. Determinants of DNA yield and quality from buccal cell samples
collected with mouthwash. Cancer Epidemiology Biomarkers & Prevention 10:
1005-1008.
Gudiseva, H.V., Hansen,
M., Gutierrez, L., Collins, D.W., He, J., Verkuil, L.D., Danford, I.D.,
Sagaser, A., Bowman, A.S., Salowe, R., Sankar, P.S., Miller-Ellis, E., Lehman,
A. & O’Brien, J.M. 2016. Saliva DNA quality and genotyping efficiency in a
predominantly elderly population. BMC Medical Genomics 9: 17.
doi:10.1186/s12920-016-0172-y.
Hansen, T.v.O.,
Simonsen, M.K., Nielsen, F.C. & Hundrup, Y.A. 2007. Collection of blood,
saliva, and buccal cell samples in a pilot study on the Danish nurse cohort:
Comparison of the response rate and quality of genomic DNA. Cancer
Epidemiology Biomarkers & Prevention 16: 2072-2076.
Heath, E.M., Morken,
N.W., Campbell, K.A., Tkach, D., Boyd, E.A. & Strom, D.A. 2001. Use of
buccal cells collected in mouthwash as a source of DNA for clinical testing. Archives
of Pathology & Laboratory Medicine 125: 127-133.
Humphrey, S.P. &
Williamson, R.T. 2001. A review of saliva: Normal composition, flow, and
function. The Journal of Prosthetic Dentistry 85: 162-169. doi:10.1067/
mpr.2001.113778.
Javaid, M.A., Ahmed,
A.S., Durand, R. & Tran, S.D. 2016. Saliva as a diagnostic tool for oral
and systemic diseases. J. Oral Biol. Craniofac. Res. 6: 66-75.
doi:10.1016/j. jobcr.2015.08.006.
King, I.B., Abouta,
J.S., Thornquist, M.D., Bigler, J., Patterson, R.E., Kristal, A.R., Shattuck,
A.L., Potter, J.D. & White, E. 2002. Buccal cell DNA yield, quality, and
collection costs comparison of methods for large-scale studies. Cancer
Epidemiology Biomarkers & Prevention 11: 1130-1133.
Koni, A.C.,
Scott, R.A., Wang, G., Bailey, M.E.S., Peplies, J., Bammann, K. &
Pitsiladis, Y.P. 2011. DNA yield and quality of saliva samples and suitability
for large-scale epidemiological studies in children. International Journal
of Obesity 35: S113-S118.
Nemoda, Z., Horvat-Gordon, M., Fortunato, C.K., Beltzer,
E.K., Scholl, J.L. & Granger, D.A. 2011. Assessing genetic polymorphisms
using DNA extracted from cells present in saliva samples. BMC Medical
Research Methodology 11: 170.
Ng, D.P., Koh, D., Choo,
S. & Chia, K.S. 2006. Saliva as a viable alternative source of human
genomic DNA in genetic epidemiology. Clinica Chimica Acta: International
Journal of Clinical Chemistry 367: 81-85. doi:10.1016/j. cca.2005.11.024.
Nunes, A.P., Oliveira,
I.O., Santos, B.R., Millech, C., Silva, L.P., González, D.A., Hallal, P.C.,
Menezes, A.M.B., Araújo, C.L. & Barros, F.C. 2012. Quality of DNA extracted
from saliva samples collected with the Oragene™ DNA self-collection kit. BMC
Medical Research Methodology 12: 65.
Osman, T.A., Costea,
D.E. & Johannessen, A.C. 2012. The use of salivary cytokines as a screening
tool for oral squamous cell carcinoma: A review of the literature. J. Oral
Maxillofac. Pathol. 16: 256-261. doi:10.4103/0973-029x.99083.
Philibert, R.A.,
Zadorozhnyaya, O., Beach, S.R. & Brody, G.H. 2008a. Comparison of the
genotyping results using DNA obtained from blood and saliva. Psychiatric
Genetics 18: 275-281. doi:10.1097/YPG.0b013e3283060f81.
Philibert, R.A.,
Zadorozhnyaya, O., Beach, S.R.H. & Brody, G.H. 2008b. A comparison of the
genotyping results using DNA obtained from blood and saliva. Psychiatric
Genetics 18: 275.
Pulford, D.J.,
Mosteller, M., Briley, J.D., Johansson, K.W. & Nelsen, A.J. 2013a. Saliva
sampling in global clinical studies: The impact of low sampling volume on
performance of DNA in downstream genotyping experiments. BMC Medical
Genomics 6: 20. doi:10.1186/1755-8794-6-20.
Pulford, D.J.,
Mosteller, M., Briley, J.D., Johansson, K.W. & Nelsen, A.J. 2013b. Saliva
sampling in global clinical studies: The impact of low sampling volume on
performance of DNA in downstream genotyping experiments. BMC Medical
Genomics 6: 20.
Robin, J.D., Ludlow,
A.T. & LaRanger, R. 2016. Comparison of DNA quantification methods for next
generation sequencing. Sci. Re. 6: 24067. doi: 10.1038/srep24067.
Sedlackova, T., Repiska,
G., Celec, P., Szemes, T. & Minarik, G. 2013. Fragmentation of DNA sffects
the accuracy of the DNA quantification by the commonly used methods. Biol.
Proced. Online. 15(1): 5. doi: 10.1186/1480-9222-15-5.
Smith, A.K., Kilaru, V.,
Klengel, T., Mercer, K.B., Bradley, B., Conneely, K.N., Ressler, K.J. &
Binder, E.B. 2015. DNA extracted from saliva for methylation studies of
psychiatric traits: Evidence tissue specificity and relatedness to brain. American
Journal of Medical Genetics. Part B, Neuropsychiatric Genetics: The Official
Publication of the International Society of Psychiatric Genetics 168b:
36-44. doi:10.1002/ajmg.b.32278.
*Pengarang untuk surat-menyurat; email: marinab@um.edu.my
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