Team:NYMU-Taipei/Project/Inhibition/Id-Nosema

From 2013.igem.org

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{{:Team:NYMU-Taipei/Header}}
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=Identifying ''Nosema ceranae''=
==Introduction==
==Introduction==
-
The identification of ''N. apis'' and ''N. ceranae'' is important and still need further research. So far the best differentiation method is the use of species–specific primers; however, the sequence differences are limited to the ribosomal RNA loci, leading to overestimation of Nosema infection rate. Thanks to recent genome study of ''N. apis'' and ''N. ceranae'', we proposed novel primers for differentiation of ''N. spp.'' based on highly conserved, species–specific protein coding genes rather than species–specific sequences in rRNA genes, which is likely to improve the accuracy of species identification.
+
The identification of ''Nosema apis'' and ''Nosema ceranae'' is important and still needs further research. So far the best differentiation method is the use of species–specific primers; however, the sequence differences are limited to the ribosomal RNA loci, leading to overestimation of Nosema infection rate. Thanks to recent genome study of ''Nosema apis'' and ''Nosema ceranae'', we proposed new primers for differentiation of ''Nosema spp.'' based on highly conserved, species–specific protein coding genes rather than species–specific sequences in rRNA genes, which is likely to improve the accuracy of species identification and can be used to quantify ''Nosema ceranae''. We confirmed the new primers by Blast, PCR with Nosema genomic DNA and the latter was sent for DNA Sequencing. After the DNA Sequencing, the result showed that there was only N. ceranae and no N. apis in the sample, while old primers produced a ''fake'' N apis band and new primers did not make the mistake.  
 +
 
==Background==
==Background==
-
===''N. spp.'' Differentiation Methods===
+
===''Nosema spp.'' Differentiation Methods===
-
*Light microscopy method: It is based on morphology difference of ''N. apis'' and ''N. ceranae'', revealing that spores of N. ceranae are oval or rod shaped like a rice, and also slightly thinner than N. apis. Light microscopy method is convenient but not scientific enough since ''N. apis'' bears a striking resemblance to ''N. ceranae''.
+
*'''Light microscopy method''': It is based on morphology difference of ''N. apis'' and ''N. ceranae'', revealing that N. ceranae are oval or rod shaped like a rice, and also slightly thinner than N. apis. Light microscopy method is convenient but not scientific enough since ''N. apis'' bears a striking resemblance to ''N. ceranae''.
{|
{|
|-
|-
-
|[[File:Nymu-Nosema.jpg|thumb|250px|'''Spores of ''N. ceranae'' and ''N. apis'' are hard to distingush under light microscope.'''<br>http://scientificbeekeeping.com/nosema-ceranae-additional-reports-and-ramblings/]]||[[File:Tem.jpg|thumb|250px|Transmission electron micrographs of (A)''N. apis'' and (B)''N. ceranae''.'''<br>http://www.zoologie.uni-halle.de/allgemeine_zoologie/research/host-parasite/]]
+
|[[File:Nymu-Nosema.jpg|thumb|250px|''' ''N. ceranae'' and ''N. apis'' are hard to distingush under light microscope.'''<br>http://scientificbeekeeping.com/nosema-ceranae-additional-reports-and-ramblings/]]||[[File:Tem.jpg|thumb|250px|Transmission electron micrographs of (A)''N. apis'' and (B)''N. ceranae''.'''<br>http://www.zoologie.uni-halle.de/allgemeine_zoologie/research/host-parasite/]]
|}
|}
{|
{|
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|}
|}
-
*Molecular methods: Species-specific primers are more accurate than light microscopy method. The problem is that so far nearly all protocols based on species–specific sequence differences are limited to the ribosomal RNA loci, which may lead to confusing results due to polymorphisms in and recombination between the multi-copy 16S rRNA genes, and hence overestimate the rate of Nosema infections.
+
*'''Molecular methods''': Species-specific primers are more accurate than light microscopy method. The problem is that so far nearly all protocols based on species–specific sequence differences are limited to the ribosomal RNA loci, which may lead to confusing results due to polymorphisms in and recombination between the multi-copy 16S rRNA genes, and hence overestimate the rate of Nosema infections.
-
[[File:Nymu-All kinds of primers.PNG|thumb|750px|center|'''Source:Molecular differentiation of Nosema apis and Nosema ceranae based on species–specific sequence differences in a protein coding gene '''<br>http://www.ncbi.nlm.nih.gov/pubmed/23352902]]
+
[[File:Nymu-All kinds of primers.PNG|thumb|750px|center|'''Source: Molecular differentiation of Nosema apis and Nosema ceranae based on species–specific sequence differences in a protein coding gene '''<br>http://www.ncbi.nlm.nih.gov/pubmed/23352902]]
-
*Our new primers: This year, we created better primers based on recently published, highly conserved and absolutely species–specific sequence differences in protein coding genes. To be more specific, the primers are based on species–specific genes instead of species–specific sequences. As more and more genes of ''Nosema spp.'' are revealed, we are able to choose the genes that are not homologs ''N. apis'' and ''N. ceranae'' for new primers. Our new primers are confirmed by BLAST through nucleotide blast and tblastn using Nucleotide collection (nr/nt) database and Whole-genome shotgun contigis (wgs) database. We propose that our new primers should become the preferred molecular tool for differentiation of ''Nosema spp.'' and thus assist in apiculture.
+
*'''Our new primers''': This year, we created new primers based on recently published, highly conserved and absolutely species–specific sequence differences in protein coding genes. To be more specific, the primers are based on species–specific genes instead of species–specific sequences. As more and more genes of ''Nosema spp.'' are revealed, we are able to choose the genes that are not homologs in ''N. apis'' and ''N. ceranae'' for new primers. Our new primers are confirmed by BLAST and PCR results and then sent for DNA Sequencing. We propose that our new primers should become the preferred molecular tool for differentiation of ''Nosema spp.'' and thus assist in apiculture.
==Experiment==
==Experiment==
-
===Primer Design===
 
-
====1.Common gene of ''N. spp.''--- NcORF-00182 hypothetical protein & NAPIS_ORF01775 chitin synthase d====
 
-
NcORF-00182
 
-
Product=1089bp 
+
===The Protocol of OldPrimers===
-
[[File:Nymu-Common ceranae.jpg|frame|center|]]
+
link to our [https://2013.igem.org/Team:NYMU-Taipei/Experiments/Protocols protocol]
-
NAPIS_ORF01775
+
====''Nosema primers''====
 +
forward primer:ggcagttatgggaagtaaca
 +
 +
reverse primer:ggtcgtcacatttcatctct
-
Product=1103bp
+
Product=208bp for Nosema
-
[[File:Nymu-Common apis.jpg|frame|center|]]
+
====''N. ceranae primers''====  
-
====2.Specific gene of ''N. ceranae''---NcORF-00714 hypothetical protein====
+
-
Product=861bp for ''N. ceranae''
+
-
[[File:Nymu-Ceranae primer.jpg|frame|center|]]
+
-
====3.Specific gene of ''N. apis''---NAPIS_ORF02138 class iv chitinase====
+
-
Product=1320bp for ''N. apis''
+
-
[[File:Apis primer.jpg|frame|center|]]
+
-
Besides testing our new primers using real PCR, we also chose traditional primers to double-check whether the bees were infected with ''N. app.''
+
-
===Traditional Primer===
+
-
[[File:Nymu-Primer test.jpg|300px|right|]]
+
-
====''N. ceranae''====  
+
forward primer:cggataaaagagtccgttacc
forward primer:cggataaaagagtccgttacc
   
   
Line 49: Line 42:
reverse primer:cacgcattgctgcatcattgac
reverse primer:cacgcattgctgcatcattgac
-
 
Product=401bp for ''N. apis''
Product=401bp for ''N. apis''
 +
{| class="wikitable"
 +
|-
 +
! Temperature !! Time
 +
|-
 +
| 94<sup>o</sup>C || 120s
 +
|-
 +
| 94<sup>o</sup>C || 30s || rowspan=3 | 30 cycles
 +
|-
 +
| 53<sup>o</sup>C || 30s
 +
|-
 +
| 72<sup>o</sup>C || 30s
 +
|-
 +
| 72<sup>o</sup>C || 600s
 +
|-
 +
|}
 +
===Results of Old Primers===
 +
[[File:Nymu old primers testing.PNG|600px|thumb|center| '''Results of Old Primers''' Electrophoresis photo 1:From the left to the right shows the Nosema genomic DNA with the new primers of ''Nosema apis'' ,''Nosema ceanae'' ,''Nosema apis'' and ''Nosema ceanae'' respectively]]
 +
The ''N. apis'' primers produced several bands rather than one, which was not coherent with the result of the previous paper; amd thus we sent the PCR mixtures for DNA Sequencing and found out that there was actually only ''N. ceranae'' and no ''N. apis'' existing in the sample. The old ''N. apis'' primers produced a fake ''N. apis'' band while our new ''N. apis'' primers did not make the mistake.   
 +
 +
===New Primers===
 +
====1. Common gene of ''N. spp.''--- NcORF_00182 hypothetical protein & NAPIS_ORF01775 chitin synthase d====
 +
NcORF_00182 for ''N. apis''
 +
 +
Product=1089bp 
 +
[[File:Nymu-Common ceranae.jpg|frame|1500px|center|]]
 +
NAPIS_ORF01775 ''N. ceranae''
 +
 +
Product=1103bp
 +
[[File:Nymu-Common apis.jpg|frame|1500px|center|]]
 +
====2. Specific gene of ''N. ceranae''---NcORF_00714 hypothetical protein====
 +
NcORF_00714
 +
 +
Product=861bp for ''N. ceranae''
 +
[[File:Nymu-Ceranae primer.jpg|frame|1500px|center|]]
 +
====3. Specific gene of ''N. apis''---NAPIS_ORF02138 class iv chitinase====
 +
NAPIS_ORF02138
 +
Product=1320bp for ''N. apis''
 +
[[File:Apis primer.jpg|frame|1500px|center|]]
 +
Besides testing our new primers, we also chose traditional primers to double-check whether the bees were infected with ''N. app.'' The first step is to do real PCR with primers and spores extracted from infected bees, and then run the electrophoresis to check the length.
 +
 +
===The Protocol of New Primers===
 +
====Nosema primers====
 +
forward primer:aatttatactggaacttttgaaaataaaagt
 +
             
 +
reverse primer:catttatttaaaatcattgtttctccacaaat
 +
             
 +
Product=1103bp for for ''N. apis''and 1089bp for ''N. ceranae''
 +
====''N. ceranae'' primers====
 +
forward primer:tcatttaattaaactttcaacgtaattttctaaattct
 +
 +
reverse primer:atgttatcacgtaatattgagaagaaacttt
 +
Product=861bp for ''N. ceranae''
 +
====''N. apis'' primers====
 +
forward primer:ttagtaacatccttcttcagaagctaaattttc
 +
 +
reverse primer:atgcaacttcaaaatccaataactactgtaac
 +
 +
Product=1320bp for ''N. apis''
 +
 +
 +
{| class="wikitable"
 +
|-
 +
! Temperature !! Time
 +
|-
 +
| 94<sup>o</sup>C || 120s
 +
|-
 +
| 94<sup>o</sup>C || 30s || rowspan=3 | 45 cycles
 +
|-
 +
| 55<sup>o</sup>C || 30s
 +
|-
 +
| 72<sup>o</sup>C || 30s
 +
|-
 +
| 72<sup>o</sup>C || 600s
 +
|-
 +
|}
 +
===Results of New Primers===
 +
[[File:Nymu-new primer result.PNG|500px|thumb|center|Electrophoresis photo 2:From the left to the right shows the Nosema genomic DNA with the new primers of ''Nosema apis'' ,''Nosema ceanae'' ,''Nosema apis'' and ''Nosema ceanae'' respectively.]]
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
-
The electrophoresis result showed that bees were infected with both ''N. apis'' and ''N. ceranae''. Another thing worth mentioning is that two bands instead of one band appeared when checking the existence of ''N. apis''. The next thing we are planning to do is to compare the outcomes of our new primers with traditional ones to verify the feasibility of the novel method.
 
==Reference==
==Reference==
Line 58: Line 155:
#Nabian, S., Ahmadi, K., Nazem, S. M., & Gerami, S. A. (January 01, 2011). First Detection of Nosema ceranae, a Microsporidian Protozoa of European Honeybees (Apis mellifera) In Iran. Iranian Journal of Parasitology, 6, 3, 89-95.[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279895/]
#Nabian, S., Ahmadi, K., Nazem, S. M., & Gerami, S. A. (January 01, 2011). First Detection of Nosema ceranae, a Microsporidian Protozoa of European Honeybees (Apis mellifera) In Iran. Iranian Journal of Parasitology, 6, 3, 89-95.[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279895/]
#Chen, Y., Pettis, J. S., Zhao, Y., Liu, X., Tallon, L. J., Sadzewicz, L. D., Li, R., ... Evans, J. D. (January 01, 2013). Genome sequencing and comparative genomics of honey bee microsporidia, Nosema apis reveal novel insights into host-parasite interactions. Bmc Genomics, 14.[http://www.biomedcentral.com/1471-2164/14/451/]   
#Chen, Y., Pettis, J. S., Zhao, Y., Liu, X., Tallon, L. J., Sadzewicz, L. D., Li, R., ... Evans, J. D. (January 01, 2013). Genome sequencing and comparative genomics of honey bee microsporidia, Nosema apis reveal novel insights into host-parasite interactions. Bmc Genomics, 14.[http://www.biomedcentral.com/1471-2164/14/451/]   
 +
#Sagastume, S., del, A. C., Martín-Hernández, R., Higes, M., & Henriques-Gil, N. (January 01, 2011). Polymorphism and recombination for rDNA in the putatively asexual microsporidian Nosema ceranae, a pathogen of honeybees. #Environmental Microbiology, 13, 1, 84-95.[http://www.ncbi.nlm.nih.gov/pubmed/21199250]
 +
#Chen, Y. P., Evans, J. D., Murphy, C., Gutell, R., Zuker, M., Gundensen-Rindal, D., & Pettis, J. S. (January 01, 2009). Morphological, molecular, and phylogenetic characterization of Nosema ceranae, a microsporidian parasite isolated from the European honey bee, Apis mellifera. The Journal of Eukaryotic Microbiology, 56, 2.)[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2779023/]
 +
#Huang, W. F., Bocquet, M., Lee, K. C., Sung, I. H., Jiang, J. H., Chen, Y. W., & Wang, C. H. (January 01, 2008). The comparison of rDNA spacer regions of Nosema ceranae isolates from different hosts and locations. Journal of Invertebrate Pathology, 97, 1, 9-13. [http://ansc.niu.edu.tw/files/writing_journal/20/27_2fcb1cab.pdf]
 +
#Bollan, K. A., Hothersall, J. D., Moffat, C., Durkacz, J., Saranzewa, N., Wright, G. A., Raine, N. E., ... Connolly, C. N. (January 01, 2013). The microsporidian parasites Nosema ceranae and Nosema apis are widespread in honeybee (Apis mellifera) colonies across Scotland. Parasitology Research, 112, 2, 751-9. [http://www.ncbi.nlm.nih.gov/pubmed/23180128]
 +
#Huang, W. F., Solter, L. F., Yau, P. M., & Imai, B. S. (January 01, 2013). Nosema ceranae escapes fumagillin control in honey bees. Plos Pathogens, 9, 3.) [http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1003185]
{{:Team:NYMU-Taipei/Footer}}
{{:Team:NYMU-Taipei/Footer}}
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#
 
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Latest revision as of 03:23, 29 October 2013

National Yang Ming University


Contents

Identifying Nosema ceranae

Introduction

The identification of Nosema apis and Nosema ceranae is important and still needs further research. So far the best differentiation method is the use of species–specific primers; however, the sequence differences are limited to the ribosomal RNA loci, leading to overestimation of Nosema infection rate. Thanks to recent genome study of Nosema apis and Nosema ceranae, we proposed new primers for differentiation of Nosema spp. based on highly conserved, species–specific protein coding genes rather than species–specific sequences in rRNA genes, which is likely to improve the accuracy of species identification and can be used to quantify Nosema ceranae. We confirmed the new primers by Blast, PCR with Nosema genomic DNA and the latter was sent for DNA Sequencing. After the DNA Sequencing, the result showed that there was only N. ceranae and no N. apis in the sample, while old primers produced a fake N apis band and new primers did not make the mistake.

Background

Nosema spp. Differentiation Methods

  • Light microscopy method: It is based on morphology difference of N. apis and N. ceranae, revealing that N. ceranae are oval or rod shaped like a rice, and also slightly thinner than N. apis. Light microscopy method is convenient but not scientific enough since N. apis bears a striking resemblance to N. ceranae.
N. ceranae and N. apis are hard to distingush under light microscope.
http://scientificbeekeeping.com/nosema-ceranae-additional-reports-and-ramblings/
Transmission electron micrographs of (A)N. apis and (B)N. ceranae.
http://www.zoologie.uni-halle.de/allgemeine_zoologie/research/host-parasite/
Some N. ceranae spores shown in 400x manification rate.
Many N. ceranae spores shown in 400x manification rate.
  • Molecular methods: Species-specific primers are more accurate than light microscopy method. The problem is that so far nearly all protocols based on species–specific sequence differences are limited to the ribosomal RNA loci, which may lead to confusing results due to polymorphisms in and recombination between the multi-copy 16S rRNA genes, and hence overestimate the rate of Nosema infections.
Source: Molecular differentiation of Nosema apis and Nosema ceranae based on species–specific sequence differences in a protein coding gene
http://www.ncbi.nlm.nih.gov/pubmed/23352902
  • Our new primers: This year, we created new primers based on recently published, highly conserved and absolutely species–specific sequence differences in protein coding genes. To be more specific, the primers are based on species–specific genes instead of species–specific sequences. As more and more genes of Nosema spp. are revealed, we are able to choose the genes that are not homologs in N. apis and N. ceranae for new primers. Our new primers are confirmed by BLAST and PCR results and then sent for DNA Sequencing. We propose that our new primers should become the preferred molecular tool for differentiation of Nosema spp. and thus assist in apiculture.


Experiment

The Protocol of OldPrimers

link to our protocol

Nosema primers

forward primer:ggcagttatgggaagtaaca

reverse primer:ggtcgtcacatttcatctct

Product=208bp for Nosema

N. ceranae primers

forward primer:cggataaaagagtccgttacc

reverse primer:tgagcagggttctagggat

Product=250bp for N. ceranae

N. apis primers

forward primer:ccattgccggataagagagt

reverse primer:cacgcattgctgcatcattgac Product=401bp for N. apis

Temperature Time
94oC 120s
94oC 30s 30 cycles
53oC 30s
72oC 30s
72oC 600s

Results of Old Primers

Results of Old Primers Electrophoresis photo 1:From the left to the right shows the Nosema genomic DNA with the new primers of Nosema apis ,Nosema ceanae ,Nosema apis and Nosema ceanae respectively

The N. apis primers produced several bands rather than one, which was not coherent with the result of the previous paper; amd thus we sent the PCR mixtures for DNA Sequencing and found out that there was actually only N. ceranae and no N. apis existing in the sample. The old N. apis primers produced a fake N. apis band while our new N. apis primers did not make the mistake.

New Primers

1. Common gene of N. spp.--- NcORF_00182 hypothetical protein & NAPIS_ORF01775 chitin synthase d

NcORF_00182 for N. apis

Product=1089bp

Nymu-Common ceranae.jpg

NAPIS_ORF01775 N. ceranae

Product=1103bp

Nymu-Common apis.jpg

2. Specific gene of N. ceranae---NcORF_00714 hypothetical protein

NcORF_00714

Product=861bp for N. ceranae

Nymu-Ceranae primer.jpg

3. Specific gene of N. apis---NAPIS_ORF02138 class iv chitinase

NAPIS_ORF02138 Product=1320bp for N. apis

Apis primer.jpg

Besides testing our new primers, we also chose traditional primers to double-check whether the bees were infected with N. app. The first step is to do real PCR with primers and spores extracted from infected bees, and then run the electrophoresis to check the length.

The Protocol of New Primers

Nosema primers

forward primer:aatttatactggaacttttgaaaataaaagt

reverse primer:catttatttaaaatcattgtttctccacaaat

Product=1103bp for for N. apisand 1089bp for N. ceranae

N. ceranae primers

forward primer:tcatttaattaaactttcaacgtaattttctaaattct

reverse primer:atgttatcacgtaatattgagaagaaacttt Product=861bp for N. ceranae

N. apis primers

forward primer:ttagtaacatccttcttcagaagctaaattttc

reverse primer:atgcaacttcaaaatccaataactactgtaac

Product=1320bp for N. apis


Temperature Time
94oC 120s
94oC 30s 45 cycles
55oC 30s
72oC 30s
72oC 600s

Results of New Primers

Electrophoresis photo 2:From the left to the right shows the Nosema genomic DNA with the new primers of Nosema apis ,Nosema ceanae ,Nosema apis and Nosema ceanae respectively.
















Reference

  1. Gisder, S., & Genersch, E. (May 01, 2013). Molecular differentiation of Nosema apis and Nosema ceranae based on species–specific sequence differences in a protein coding gene. Journal of Invertebrate Pathology, 113, 1, 1-6.[http://www.sciencedirect.com/science/article/pii/S0022201113000165]
  2. Nabian, S., Ahmadi, K., Nazem, S. M., & Gerami, S. A. (January 01, 2011). First Detection of Nosema ceranae, a Microsporidian Protozoa of European Honeybees (Apis mellifera) In Iran. Iranian Journal of Parasitology, 6, 3, 89-95.[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279895/]
  3. Chen, Y., Pettis, J. S., Zhao, Y., Liu, X., Tallon, L. J., Sadzewicz, L. D., Li, R., ... Evans, J. D. (January 01, 2013). Genome sequencing and comparative genomics of honey bee microsporidia, Nosema apis reveal novel insights into host-parasite interactions. Bmc Genomics, 14.[http://www.biomedcentral.com/1471-2164/14/451/]
  4. Sagastume, S., del, A. C., Martín-Hernández, R., Higes, M., & Henriques-Gil, N. (January 01, 2011). Polymorphism and recombination for rDNA in the putatively asexual microsporidian Nosema ceranae, a pathogen of honeybees. #Environmental Microbiology, 13, 1, 84-95.[http://www.ncbi.nlm.nih.gov/pubmed/21199250]
  5. Chen, Y. P., Evans, J. D., Murphy, C., Gutell, R., Zuker, M., Gundensen-Rindal, D., & Pettis, J. S. (January 01, 2009). Morphological, molecular, and phylogenetic characterization of Nosema ceranae, a microsporidian parasite isolated from the European honey bee, Apis mellifera. The Journal of Eukaryotic Microbiology, 56, 2.)[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2779023/]
  6. Huang, W. F., Bocquet, M., Lee, K. C., Sung, I. H., Jiang, J. H., Chen, Y. W., & Wang, C. H. (January 01, 2008). The comparison of rDNA spacer regions of Nosema ceranae isolates from different hosts and locations. Journal of Invertebrate Pathology, 97, 1, 9-13. [http://ansc.niu.edu.tw/files/writing_journal/20/27_2fcb1cab.pdf]
  7. Bollan, K. A., Hothersall, J. D., Moffat, C., Durkacz, J., Saranzewa, N., Wright, G. A., Raine, N. E., ... Connolly, C. N. (January 01, 2013). The microsporidian parasites Nosema ceranae and Nosema apis are widespread in honeybee (Apis mellifera) colonies across Scotland. Parasitology Research, 112, 2, 751-9. [http://www.ncbi.nlm.nih.gov/pubmed/23180128]
  8. Huang, W. F., Solter, L. F., Yau, P. M., & Imai, B. S. (January 01, 2013). Nosema ceranae escapes fumagillin control in honey bees. Plos Pathogens, 9, 3.) [http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1003185]

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