مقایسه حساسیت ایمونوسنسور مبتنی بر نانوذرات سیلیکای رنگی با روش کشت و واکنش زنجیره ای پلی مراز در تشخیص بروسلا آبورتوس
محورهای موضوعی : زیست فناوری میکروبیآرش شمس 1 , بهاره رحیمیان ظریف 2 , مجتبی صلوتی 3 , رضا شاپوری 4 , ساکو میرزایى 5
1 - دانشجوی دکتری، دانشگاه آزاد اسلامی، واحد سنندج، دانشکده علوم پایه، گروه بیوشیمی
2 - استادیار، دانشگاه آزاد اسلامی، واحد سنندج، دانشکده علوم پایه، گروه میکروبیولوژی
3 - استاد، دانشگاه آزاد اسلامی، واحد زنجان، دانشکده علوم پایه، مرکز تحقیقات بیولوژی
4 - استادیار، دانشگاه آزاد اسلامی، واحد زنجان، دانشکده علوم پایه، گروه میکروبیولوژی
5 - استادیار، دانشگاه آزاد اسلامی، واحد سنندج، دانشکده علوم پایه، گروه بیوشیمی
کلید واژه: PCR, کشت, بروسلا آبورتوس, ایمونوسنسور, سیلیکای رنگی,
چکیده مقاله :
سابقه و هدف: بروسلوز، همواره به عنوان تهدیدی بر سلامت و اقتصاد جامعه مطرح بوده است. وجود محدودیت در روش های تشخیص بیماری در انسان و حیوان لزوم استفاده از روش های نوین شناسایی را توجیه می کند. این مطالعه با هدف مقایسه بین میزان حساسیت روش کشت و PCR با ایمونوسنسور با استفاده از راهبرد رنگ سنجی به منظور تشخیص بروسلا آبورتوس انجام شد.مواد و روش‌ها: نانوذرات سیلیکای رنگی و نانوذرات پارامغناطیس پس از سنتز، با آنتی بادی پلی کلونال ضد بروسلا آبورتوس فعال شده با کمپلکس EDC/NHS کونژوگه گردیدند تا به ترتیب پروب های شناساگر و تسخیری تشکیل گردند. این پروب ها پس از اضافه شدن به رقت های سریالی از بروسلا آبورتوس و تکمیل واکنش، جمع آوری شده و در ادامه، با رها سازی رنگ آلی از ساختار سیلیکا شدت جذب در670 نانومتر قرائت گردید. از سوی دیگر، هم زمان با کشت هر رقت، DNA کروموزومی مربوط به آن توسط کیت استخراج و آزمون PCR انجام گرفت. نتایج هرسه آزمون در نهایت با یکدیگر مقایسه شد.یافته‌ها: بر اساس مشاهدات، دامنه تشخیص در ایمونوسنسور و کشت یکسان و برابر CFU.ml-1 108×1.5 - 103×1.5 گزارش گردید. حداقل میزان تشخیص در ایمونوسنسور CFU.ml-1450 و در کشت CFU.ml-1400 تعیین شد. دامنه تشخیص PCR نیز CFU.ml-1 108×1.5 - 104×1.5 با حداقل میزان تشخیص CFU.mL-1 5000 به دست آمد.نتیجه گیری: مقایسه نتایج این تحقیق نشان داد که ایمونوسنسور پیشنهادی قابلیت جایگزینی روش های مرسوم شناسایی بروسلا آبورتوس را دارد و می تواند به عنوان یک ابزار تشخیص در محل با داشتن حساسیت بالا مطرح گردد.
Background & Objectives: Brucellosis has always been a threat to the health and economy of the community. The limitations of human and animal disease detection methods justify the need for new diagnostic methods. This study was aimed to compare the sensitivity of colorimetric strategy based- immunosensors in the diagnosis of Brucella abortus with those of culturing and PCR methods. Materials & Methods: The colored silica nanoparticles and paramagnetic nanoparticles after synthesis were conjugated with polyclonal antibody to form EDC/NHS complexes to form probe sequences, respectively. These probes were collected after being added to serial dilutions of B. abortus and completing the reaction. Then, with releasing organic dye from the silica structure, absorbance intensity was measured at 670 nm. On the other hand, at the time of each dilution, the corresponding chromosomal DNA was extracted by a DNA extraction kit and used for PCR analysis. The results of all three tests were ultimately compared. Results: Based on the results, the detection range of the immunosensor and culture was the same and equal to 1.5×103 - 1.5×108 CFU mL-1. But, the limit of detection for immunosensor and culture was measured as 450 CFU mL-1 and 400 CFU mL-1, respectively. The results of the PCR test exhibited a wide dynamic range of 1.5×104 to1.5×108 CFU mL-1, with LOD of 5000 CFU mL-1.Conclusion: Comparing the results of this study showed that the proposed immunosensor is capable of replacing conventional B. abortus detection methods and can be considered as an on-site diagnostic tool, with high sensitivity.
enumeration of colony count in live Brucella vaccines. J Veterinary World. 2017; 10(6):
610-615.
2. Godfroid J. Brucellosis in livestock and wildlife: zoonotic diseases without pandemic potential
in need of innovative one health approaches. Arch Public Health. 2017; 75(1): 34.
3. Minda A, Gezahegne MK. A review on diagnostic methods of brucellosis. Veterinar Sci Tech.
2016; 7: 3.
4. Raghava S, Umesha S. Brucellosis a review on the diagnostic techniques and medical plants
used in the management of the brucellosis. J World Pharmacy Pharmaceutical Sci. 2018; 7(6):
2278-4357.
5. Christopher S, Umapathy B, Ravikumar K. Brucellosis: review on the recent trends in
pathogenicity and laboratory diagnosis. J Lab Physic. 2010; 2(2): 55.
6. Santis Rd. Brucella: molecular diagnostic techniques in response to bioterrorism threat. J
Bioterror Biodefense. 2014; 5(14): 20.
7. Sun Q, Zhao G, Dou W. An optical and rapid sandwich immunoassay method for detection of
Salmonella pullorum and Salmonella gallinarum based on immune blue silica nanoparticles
and magnetic nanoparticles. Sensor Actuator B-Chem. 2016; 226: 69-75.
8. Sun Q, Zhao G, Dou w. Blue silica nanoparticle-based colorimetric immunoassay for detection
of Salmonella pullorum. Anal Methods. 2015; 7(20): 8647-8654.
9. Sun Q, Zhao G, Dou W. A nonenzymatic optical immunoassay strategy for detection of
Salmonella infection based on blue silica nanoparticles. Anal Chim Acta. 2015; 898: 109-115.
10. Piriya VSA, Joseph P, Daniel K, Lakshmanan S, Kinoshita T, Muthusamy S. Colorimetric
sensors for rapid detection of various analytes. Materials Sci Eng. 2017; 78: 1231-1245.
11. Koźlecki T. Improved synthesis of nanosized silica in water-in-oil microemulsions. J
Nanoparticles. 2016; 3(2): 205.
12. Zhu C, Zhao G, Dou W. A new synthesis method for bright monodispersed core-shell colored
silica submicron particles. J Sol Gel Sci Tech. 2018; 85(1): 76-83.
13. Mohapatra M, Anand S. Synthesis and applications of nano-structured iron oxides/hydroxides,
a review. Int J Eng Sci Tech. 2010; 2(8): 268-275.
14. Wei Y. Synthesis of Fe3O4 nanoparticles and their magnetic properties. Procedia Eng J. 2012;
27: 632-637.
15. Bagwe RP. Optimization of dye-doped silica nanoparticles prepared using a reverse
microemulsion method. Langmuir. 2004; 20(19): 8336-8342.
16. Amini B. Fluorescence bio-barcode DNA assay based on gold and magnetic nanoparticles for
detection of Exotoxin A gene sequence. Biosens Bioelectron. 2017; 92: 679-686.
17. Yu H, Zhao G, Dou W. Simultaneous detection of pathogenic bacteria using agglutination test
based on colored silica nanoparticles. CurrPharm Biotechnol. 2015; 16(8): 716-723.
18. Siadat SD. Preparation and evaluation of a new lipopolysaccharide-based conjugate as a
vaccine candidate for brucellosis. Osong Public Health Res Perspectives. 2015; 6(1): 9-13.
19. Shams N, Jaidari A, Etemadfar L. Molecular detection of Brucella abortus and Brucella
melitensis in raw and unpasteurized bulk cow milk tanks of traditional domestic dairy sale
centers in Khorramabad. Iranian J Med Microbiol. 2017; 11(4): 13-20.
20. Azizi F, Hatami H, Janghorbani M. Epidemiology and control of common diseases in Iran.
Tehran: Eshtiagh Publications. 2000; 602-616.
21. Alavi SM. Brucellosis risk factors in the southwestern province of Khuzestan, Iran. Int J
Enteric Pathog. 2014; 2(1): 1-4.
22. Roushan MRH, Ebrahimpour S. Human brucellosis: An overview. Caspian J Med. 2015;
6(1): 46.
23. Šiširak M, Hukić M, Knežević Z. Evaluation of some diagnostic methods for the brucellosis in
humans-a five-year study. Prilozi. 2010; 31(1): 91-101.
_||_
enumeration of colony count in live Brucella vaccines. J Veterinary World. 2017; 10(6):
610-615.
2. Godfroid J. Brucellosis in livestock and wildlife: zoonotic diseases without pandemic potential
in need of innovative one health approaches. Arch Public Health. 2017; 75(1): 34.
3. Minda A, Gezahegne MK. A review on diagnostic methods of brucellosis. Veterinar Sci Tech.
2016; 7: 3.
4. Raghava S, Umesha S. Brucellosis a review on the diagnostic techniques and medical plants
used in the management of the brucellosis. J World Pharmacy Pharmaceutical Sci. 2018; 7(6):
2278-4357.
5. Christopher S, Umapathy B, Ravikumar K. Brucellosis: review on the recent trends in
pathogenicity and laboratory diagnosis. J Lab Physic. 2010; 2(2): 55.
6. Santis Rd. Brucella: molecular diagnostic techniques in response to bioterrorism threat. J
Bioterror Biodefense. 2014; 5(14): 20.
7. Sun Q, Zhao G, Dou W. An optical and rapid sandwich immunoassay method for detection of
Salmonella pullorum and Salmonella gallinarum based on immune blue silica nanoparticles
and magnetic nanoparticles. Sensor Actuator B-Chem. 2016; 226: 69-75.
8. Sun Q, Zhao G, Dou w. Blue silica nanoparticle-based colorimetric immunoassay for detection
of Salmonella pullorum. Anal Methods. 2015; 7(20): 8647-8654.
9. Sun Q, Zhao G, Dou W. A nonenzymatic optical immunoassay strategy for detection of
Salmonella infection based on blue silica nanoparticles. Anal Chim Acta. 2015; 898: 109-115.
10. Piriya VSA, Joseph P, Daniel K, Lakshmanan S, Kinoshita T, Muthusamy S. Colorimetric
sensors for rapid detection of various analytes. Materials Sci Eng. 2017; 78: 1231-1245.
11. Koźlecki T. Improved synthesis of nanosized silica in water-in-oil microemulsions. J
Nanoparticles. 2016; 3(2): 205.
12. Zhu C, Zhao G, Dou W. A new synthesis method for bright monodispersed core-shell colored
silica submicron particles. J Sol Gel Sci Tech. 2018; 85(1): 76-83.
13. Mohapatra M, Anand S. Synthesis and applications of nano-structured iron oxides/hydroxides,
a review. Int J Eng Sci Tech. 2010; 2(8): 268-275.
14. Wei Y. Synthesis of Fe3O4 nanoparticles and their magnetic properties. Procedia Eng J. 2012;
27: 632-637.
15. Bagwe RP. Optimization of dye-doped silica nanoparticles prepared using a reverse
microemulsion method. Langmuir. 2004; 20(19): 8336-8342.
16. Amini B. Fluorescence bio-barcode DNA assay based on gold and magnetic nanoparticles for
detection of Exotoxin A gene sequence. Biosens Bioelectron. 2017; 92: 679-686.
17. Yu H, Zhao G, Dou W. Simultaneous detection of pathogenic bacteria using agglutination test
based on colored silica nanoparticles. CurrPharm Biotechnol. 2015; 16(8): 716-723.
18. Siadat SD. Preparation and evaluation of a new lipopolysaccharide-based conjugate as a
vaccine candidate for brucellosis. Osong Public Health Res Perspectives. 2015; 6(1): 9-13.
19. Shams N, Jaidari A, Etemadfar L. Molecular detection of Brucella abortus and Brucella
melitensis in raw and unpasteurized bulk cow milk tanks of traditional domestic dairy sale
centers in Khorramabad. Iranian J Med Microbiol. 2017; 11(4): 13-20.
20. Azizi F, Hatami H, Janghorbani M. Epidemiology and control of common diseases in Iran.
Tehran: Eshtiagh Publications. 2000; 602-616.
21. Alavi SM. Brucellosis risk factors in the southwestern province of Khuzestan, Iran. Int J
Enteric Pathog. 2014; 2(1): 1-4.
22. Roushan MRH, Ebrahimpour S. Human brucellosis: An overview. Caspian J Med. 2015;
6(1): 46.
23. Šiširak M, Hukić M, Knežević Z. Evaluation of some diagnostic methods for the brucellosis in
humans-a five-year study. Prilozi. 2010; 31(1): 91-101.