آنالیز منحنی ذوب با شفافیت بالا(HRM) به منظور شناسایی سریع و دقیق باکتری سالمونلا بااستفاده از ژن invA
محورهای موضوعی : میکروب شناسی تشخیصیحسن نیلی 1 , سید علی قرشی 2 , حبیب ا.. دادرس 3 , محمد صادق سعید ابادی 4
1 - گروه علوم درمانگاهی دانشکده دامپزشکی دانشگاه شیراز
2 - School of Animal and Veterinary Sciences , Charles Sturt University , Wagga Wagga , Australia.
3 - School of Veterinary Medicine , Shiraz University , Shiraz , Iran.
4 - استادیار، سازمان تحقیقات و آموزش و ترویج کشاورزی، موسسه تحقیقات واکسن و سرم سازی رازی، بخش تحقیقات بیماریهای طیور
کلید واژه: ژن invA, بیماری عفونی, روش جدید تشخیص,
چکیده مقاله :
سابقه و هدف: سالمونلوز یکی از بیماریهای عفونی و مشترک بین انسان و حیوانات است که به وسیله سویههای مختلف سالمونلا ایجاد می گردد. پیشرفت در روشهای تشخیص مولکولی، شناسایی پاتوژنهای غذایی را دقیق تر و راحت کرده است. هدف از این پژوهش استفاده از روش HRM به منظور شناسایی سریع و دقیق باکتری سالمونلا با استفاده از ژن inv A میباشد.مواد و روش ها: در این تحقیق شناسایی باکتری سالمونلا با واکنش زنجیره ای پلیمراز با روش منحنی ذوب با شفافیت بالا ( PCR-HRM) به وسیله توالی ژن invA، به عنوان یک نشانگرانجام شد.در مجموع تعداد 9 سویه رفرانس سالمونلا با استفاده از یک جفت آغازگراختصاصی ژن invA برای تشخیص باکتری سالمونلا استفاده شدند.یافته ها: قطعات موردانتظار 284 جفت بازی پس از تکثیر ژنinvA توسط PCR به دست آمد. تمام نمونه های مورد آزمون قادر به تولید پیک منحنی ذوب اختصاصی سالمونلا با شفافیت بالا در بازه دمایی 87/8 تا 87/9 درجه سلسیوس بودند.نتیجه گیری:نتایج نشان داد که روش شفافیت منحنی ذوب با استفاده از آغازگر های اختصاصی ژن invA می تواند به عنوان یک روش دقیق و مطمئن به منظور تعیین جنسهای سالمونلا مورد استفاده قرار گیرد.
Background & Objectives: Salmonellosis is an infectious and common disease between humans and animals that is caused by different strains of Salmonella. Progress in molecular diagnostic methods, has led to accurate and easy detection and characterization of food microbial agent. The purpose of this research was to use HRM technique to access more accurate and rapid diagnosis of Salmonella bacteria by means of invA gene. Material & Methods: In this study, diagnosis of Salmonella was done by polymerase chain reaction and high resolution melting curve ( PCR-HRM) using the sequence on invasion A gene (invA) as a marker. In total 9 Salmonella reference strains using a specific primer pair of genes invA were used to detect Salmonella. Results: The expected size of PCR amplified fragments of invA gene was determined as 284bp. All tested strains were able to show a Salmonella specific melting curve with high resolution at thermal interval of 87.8-87.9°C. Conclusion: The results showed that HRM using specific primers of invA gene can be used as an accurate and reliable technique for diagnosis Genus of Salmonella.
virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination
assay. Journal Clin Microbiol. 199 ; 34(10): 2 19-2 22.
2. Nair S, Patel V, Hickey T, Maguire C, Greig DR, Lee W, Godbole G, Grant K, Chattaway MA.
Real-time PCR assay for differentiation of typhoidal and nontyphoidal Salmonella. J Clin
Microbiol. 2019; 7(8): e001 7-19.
3. Hyeon J- , Mann DA, Wang J, Kim WK, Deng . Rapid detection of Salmonella in poultry
environmental samples using real-time PCR coupled with immunomagnetic separation and
whole genome amplification. Poultry Sci. 2019; 98(12): 973- 979.
4. Darushi M, Doosti A, Kargar M. The prevalence of plasmid genes spv , spvC and spvR in
Salmonella enteritidis isolated from poultry industry in Chaharmahal va akhtiari Province. J
Microb World. 201 ; 7(4): 281-288.
5. Shah DH, Park J-H, Cho M-R, Kim M-C, Chae J-S. Allele-specific PCR method based on rfbS
sequence for distinguishing Salmonella gallinarum from Salmonella ullorum:
serotype-specific rfbS sequence polymorphism. J of Microbiolo Meth. 200 ; 0(2): 1 9-177.
6. ai M, Wang C, in H, Tian , Li J. Evaluation of different reaction systems for HRM
analysis in apple. iosc Meth. 2012; 3.
7. Du ois CL, Dubois DA. Strategic HRM as social design for environmental sustainability in
organization. Hum Res Manag. 2012; 1( ): 799-82 .
8. Slinger R, ellfoy D, Desjardins M, Chan . High-resolution melting assay for the detection of
gyrA mutations causing quinolone resistance in Salmonella enterica serovars Typhi and
Paratyphi. Diagn Microbiol Infect Dis. 2007; 7(4): 4 -4 8.
9. Rahn K, De Grandis SA, Clarke RC, McEwen SA, Galan JE, Ginocchio C. Amplification of an
invA gene sequence of Salmonella ty himurium by polymerase chain reaction as a specific
method of detection of Salmonella. Mol Cellul Probes. 1992; (4): 271-279.
10. Sanders K, Shipton H, Gomes J . Guest editors introduction: Is the HRM process
important? Past, current, and future challenges. Hum Res Manag. 2014; 3(4): 489- 03.
11. ingga G, Liu Z, Zhang J, Zhu , Lin L, Ding S. High resolution melting curve analysis as
a new tool for rapid identification of canine parvovirus type 2 strains. Mol Cellul Probes. 2014;
28( - ): 271-278.
12. Ren , u , u C, eng Z, Li M, Zhang L. High resolution melting (HRM) analysis as a
new tool for rapid identification of Salmonella enterica serovar Gallinarum biovars Pullorum
and Gallinarum. Poultry Sci. 201 ; 9 ( ): 1088-1093.
13. Hoseinpour M, Sabokbar A, aklhtiari A, Parsa S. Comparison of bacterial culture, ELISA
and PCR techniques for detection of Salmonella in poultry meat samples collected from
Tehran. J Microb World. 2013; ( ): 2-72.
14. ell RL, Jarvis KG, Ottesen AR, Mc arland MA, rown EW. Recent and emerging
innovations in Salmonella detection: a food and environmental perspective. Microb
iotechnol. 201 ; 9(3): 279-292.
15. Rahn K, De Grandis S, Clarke R, McEwen S, Galan J, Ginocchio C. Amplification of an
invA gene sequence of Salmonella ty himurium by polymerase chain reaction as a specific
method of detection of Salmonella. Mol Cellul Probes. 1992; (4): 271-279.
16. Galan JE, Ginocchio C, Costeas P. Molecular and functional characterization of the
Salmonella invasion gene invA: homology of InvA to members of a new protein family. J
acteriol. 1992; 174(13): 4338-4349.
17. Nucera DM, Maddox CW, Hoien-Dalen P, Weigel RM. Comparison of API 20E and invA
PCR for identification of Salmonella enterica isolates from swine production units. J Clin
Microbiol. 200 ; 44(9): 3388-3390.
18. Salehi TZ, Mahzounieh M, Saeedzadeh A. Detection of invA gene in isolated Salmonella
from broilers by PCR method. Int J Poult Sci. 200 ; 4(8): 7- 9.
19. Zambounis A, Ganopoulos I, Chatzidimopoulos M, Tsaftaris A, Madesis P. High-resolution
melting approaches towards plant fungal molecular diagnostics. Phytoparasitica. 201 ; 43(2):
2 -272.
20. Diaz MH, Winchell JM. The evolution of advanced molecular diagnostics for the detection
and characterization of Mycoplasma pneumoniae. ront Microbiol. 201 ; 7: 232-242.
21. Hu M, ang D, Wu , Luo M, u . A novel high-resolution melting analysis-based
method for Salmonella genotyping. J Microbiol Meth. 2020; 172: 10 -110.
22. ratchikov M, Mauricas M. Development of a multiple-run high-resolution melting assay
for Salmonella spp. genotyping: HRM application for Salmonella spp. subtyping. Diagn
Microbiol Infect Dis. 2011; 71(3): 192-200.
23. Jeng K, ang S, Won H, Gaydos CA, Hsieh -H, Kecojevic A, Carroll KC, Hardick J,
Rothman RE. Application of a 1 S rRNA PCR–high-resolution melt analysis assay for rapid
detection of Salmonella bacteremia. J Clin Microbiol. 2012; 0(3): 1122-1124.
24. Syropoulou , Parlapani , osmali I, Madesis P, oziaris IS. HRM and 1 S rRNA gene
sequencing reveal the cultivable microbiota of the European sea bass during ice storage.
International J ood Microbiol. 2020; 327: 108-11 .
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virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination
assay. Journal Clin Microbiol. 199 ; 34(10): 2 19-2 22.
2. Nair S, Patel V, Hickey T, Maguire C, Greig DR, Lee W, Godbole G, Grant K, Chattaway MA.
Real-time PCR assay for differentiation of typhoidal and nontyphoidal Salmonella. J Clin
Microbiol. 2019; 7(8): e001 7-19.
3. Hyeon J- , Mann DA, Wang J, Kim WK, Deng . Rapid detection of Salmonella in poultry
environmental samples using real-time PCR coupled with immunomagnetic separation and
whole genome amplification. Poultry Sci. 2019; 98(12): 973- 979.
4. Darushi M, Doosti A, Kargar M. The prevalence of plasmid genes spv , spvC and spvR in
Salmonella enteritidis isolated from poultry industry in Chaharmahal va akhtiari Province. J
Microb World. 201 ; 7(4): 281-288.
5. Shah DH, Park J-H, Cho M-R, Kim M-C, Chae J-S. Allele-specific PCR method based on rfbS
sequence for distinguishing Salmonella gallinarum from Salmonella ullorum:
serotype-specific rfbS sequence polymorphism. J of Microbiolo Meth. 200 ; 0(2): 1 9-177.
6. ai M, Wang C, in H, Tian , Li J. Evaluation of different reaction systems for HRM
analysis in apple. iosc Meth. 2012; 3.
7. Du ois CL, Dubois DA. Strategic HRM as social design for environmental sustainability in
organization. Hum Res Manag. 2012; 1( ): 799-82 .
8. Slinger R, ellfoy D, Desjardins M, Chan . High-resolution melting assay for the detection of
gyrA mutations causing quinolone resistance in Salmonella enterica serovars Typhi and
Paratyphi. Diagn Microbiol Infect Dis. 2007; 7(4): 4 -4 8.
9. Rahn K, De Grandis SA, Clarke RC, McEwen SA, Galan JE, Ginocchio C. Amplification of an
invA gene sequence of Salmonella ty himurium by polymerase chain reaction as a specific
method of detection of Salmonella. Mol Cellul Probes. 1992; (4): 271-279.
10. Sanders K, Shipton H, Gomes J . Guest editors introduction: Is the HRM process
important? Past, current, and future challenges. Hum Res Manag. 2014; 3(4): 489- 03.
11. ingga G, Liu Z, Zhang J, Zhu , Lin L, Ding S. High resolution melting curve analysis as
a new tool for rapid identification of canine parvovirus type 2 strains. Mol Cellul Probes. 2014;
28( - ): 271-278.
12. Ren , u , u C, eng Z, Li M, Zhang L. High resolution melting (HRM) analysis as a
new tool for rapid identification of Salmonella enterica serovar Gallinarum biovars Pullorum
and Gallinarum. Poultry Sci. 201 ; 9 ( ): 1088-1093.
13. Hoseinpour M, Sabokbar A, aklhtiari A, Parsa S. Comparison of bacterial culture, ELISA
and PCR techniques for detection of Salmonella in poultry meat samples collected from
Tehran. J Microb World. 2013; ( ): 2-72.
14. ell RL, Jarvis KG, Ottesen AR, Mc arland MA, rown EW. Recent and emerging
innovations in Salmonella detection: a food and environmental perspective. Microb
iotechnol. 201 ; 9(3): 279-292.
15. Rahn K, De Grandis S, Clarke R, McEwen S, Galan J, Ginocchio C. Amplification of an
invA gene sequence of Salmonella ty himurium by polymerase chain reaction as a specific
method of detection of Salmonella. Mol Cellul Probes. 1992; (4): 271-279.
16. Galan JE, Ginocchio C, Costeas P. Molecular and functional characterization of the
Salmonella invasion gene invA: homology of InvA to members of a new protein family. J
acteriol. 1992; 174(13): 4338-4349.
17. Nucera DM, Maddox CW, Hoien-Dalen P, Weigel RM. Comparison of API 20E and invA
PCR for identification of Salmonella enterica isolates from swine production units. J Clin
Microbiol. 200 ; 44(9): 3388-3390.
18. Salehi TZ, Mahzounieh M, Saeedzadeh A. Detection of invA gene in isolated Salmonella
from broilers by PCR method. Int J Poult Sci. 200 ; 4(8): 7- 9.
19. Zambounis A, Ganopoulos I, Chatzidimopoulos M, Tsaftaris A, Madesis P. High-resolution
melting approaches towards plant fungal molecular diagnostics. Phytoparasitica. 201 ; 43(2):
2 -272.
20. Diaz MH, Winchell JM. The evolution of advanced molecular diagnostics for the detection
and characterization of Mycoplasma pneumoniae. ront Microbiol. 201 ; 7: 232-242.
21. Hu M, ang D, Wu , Luo M, u . A novel high-resolution melting analysis-based
method for Salmonella genotyping. J Microbiol Meth. 2020; 172: 10 -110.
22. ratchikov M, Mauricas M. Development of a multiple-run high-resolution melting assay
for Salmonella spp. genotyping: HRM application for Salmonella spp. subtyping. Diagn
Microbiol Infect Dis. 2011; 71(3): 192-200.
23. Jeng K, ang S, Won H, Gaydos CA, Hsieh -H, Kecojevic A, Carroll KC, Hardick J,
Rothman RE. Application of a 1 S rRNA PCR–high-resolution melt analysis assay for rapid
detection of Salmonella bacteremia. J Clin Microbiol. 2012; 0(3): 1122-1124.
24. Syropoulou , Parlapani , osmali I, Madesis P, oziaris IS. HRM and 1 S rRNA gene
sequencing reveal the cultivable microbiota of the European sea bass during ice storage.
International J ood Microbiol. 2020; 327: 108-11 .