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Chapter 4 : Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia
Authors:
Louis M. Weiss1,
Charles R. Vossbrinck2
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Affiliations:
1: Albert Einstein College of Medicine, 1300 Morris Park Avenue, Room 504 Forchheimer, Bronx, NY 10461;
2: The Connecticut Agricultural Experiment Station, 123 Huntington Street, New Haven, CT 06504, and Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461
Content Type: Monograph
Publication Year:
1999
Category: Fungi and Fungal Pathogenesis
Book DOI:
10.1128/9781555818227
Chapter DOI:
10.1128/9781555818227.ch4
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Abstract:
The recognition of microsporidia as opportunistic pathogens in humans has led to increased interest in their molecular biology. Much of the recent work has focused on determination of the nucleotide sequences for rRNA genes. These sequences have been used in the development of diagnostic tools for species identification. In addition, such rRNA gene sequences have facilitated the development of a molecular phylogeny of these organisms. Given the increasing evidence of the importance of the microsporidia as both human and agricultural pathogens, recent work has focused on the identification of microsporidial genes that could serve as potential therapeutic targets. Prior to the advent of comparative analysis of molecular data, the classification of eukaryotes was based on morphological, ecological, and physiological characteristics. Speculation based on this information is problematic because the homology among such characters is often not discernible and characters that could be considered were thought to be either shared or derived depending on the underlying hypothesis used in construction of the phylogeny. A much different view of the phylogenetic placement of the Microspora has been suggested based on analysis of β-tubulin genes. The molecular data present an excellent means of identifying a species and provide an excellent data set for proposing evolutionary relatedness through phylogenetic analysis. Molecular techniques for the diagnosis of microsporidiosis appear to have high sensitivity and specificity. These methods have proven extremely useful both in the identification of animal models as well as in investigations of the epidemiology of microsporidia pathogenic to humans.
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
Figures
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia
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Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.fig4-1
FIGURE 1
PCR of E. cuniculi with the microsporidian rRNA primers described in Table 2 . Lane S, standard (50, 100, 200, 300, 400, 500, 700, 1,000, 1,500, and 2,000 bp); lane 1, 18f::350r; lane 2, 18f::530r; lane 3, 18f:;1047r; lane 4, 18f::1492r;lane 5 , 18f-212rl; lane 6, 350f-1492r; lane 7, 530f::1492r; lane 8, 1061f-1492r; lane 9, 530f- 212rl . Products of the predicted sizes are present w i t h all t h e primer sets. In cases in which more than one band is present, cloning of the amplicon of the correct size usually yields the rRNA fragment of interest. Additional bands are due to hybridization to unrelated genes in the samples.
10.1128/9781555818227/fig4-1_thmb.gif
10.1128/9781555818227/fig4-1.gif
FIGURE 1
PCR of E. cuniculi with the microsporidian rRNA primers described in Table 2 . Lane S, standard (50, 100, 200, 300, 400, 500, 700, 1,000, 1,500, and 2,000 bp); lane 1, 18f::350r; lane 2, 18f::530r; lane 3, 18f:;1047r; lane 4, 18f::1492r;lane 5 , 18f-212rl; lane 6, 350f-1492r; lane 7, 530f::1492r; lane 8, 1061f-1492r; lane 9, 530f- 212rl . Products of the predicted sizes are present w i t h all t h e primer sets. In cases in which more than one band is present, cloning of the amplicon of the correct size usually yields the rRNA fragment of interest. Additional bands are due to hybridization to unrelated genes in the samples.
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.fig4-2
FIGURE 2
Multikingdom phylogeny inferred from 16S rRNA sequences.The phylogenetic tree was inferred by using unambiguously aligned sequences with the neighbor joining method in the PHYLIP program. Bootstrap support for topological elements in the tree is based on 200 resamplings. Horizontal distances between nodes of the tree represent relative evolutionary distances. The bar scale corresponds to 10 changes per 100 positions. (Reprinted with permission from Leipe et al., 1993. )
10.1128/9781555818227/fig4-2_thmb.gif
10.1128/9781555818227/fig4-2.gif
FIGURE 2
Multikingdom phylogeny inferred from 16S rRNA sequences.The phylogenetic tree was inferred by using unambiguously aligned sequences with the neighbor joining method in the PHYLIP program. Bootstrap support for topological elements in the tree is based on 200 resamplings. Horizontal distances between nodes of the tree represent relative evolutionary distances. The bar scale corresponds to 10 changes per 100 positions. (Reprinted with permission from Leipe et al., 1993. )
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.fig4-3
FIGURE 3
EF-lα tree of e u karyotes with archaebacteria as an outgroup. The phylogenetic tree was constructed by the maximun likelihood method based on the JTT model. The horizontal length of each branch is proportional to the estimated number of substitutions. Bootstrap probabilities are shown in parentheses. (Reprinted with permission from Kamaishi et al., 1996b. )
10.1128/9781555818227/fig4-3_thmb.gif
10.1128/9781555818227/fig4-3.gif
FIGURE 3
EF-lα tree of e u karyotes with archaebacteria as an outgroup. The phylogenetic tree was constructed by the maximun likelihood method based on the JTT model. The horizontal length of each branch is proportional to the estimated number of substitutions. Bootstrap probabilities are shown in parentheses. (Reprinted with permission from Kamaishi et al., 1996b. )
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.fig4-4
FIGURE 4
Phylogenetic tree of representative partial β-tubulin sequences (residues 108 to 259). Selected α-tubulin and γ-tubulin sequences are included, with S. pombe α-tubulin as the designated outgroup. Parsimony analysis (100 bootstrap resamplings) was used for the consensus tree. Numbers are percentages of trees in which the group of species to the right of that branch was found. A nearly identical tree, except that E. cuniculi and S. commune branches were reversed, was obtained with a distance matrix method (500 resamplings). (Reprinted with permission from Edlind et al.,1996. )
10.1128/9781555818227/fig4-4_thmb.gif
10.1128/9781555818227/fig4-4.gif
FIGURE 4
Phylogenetic tree of representative partial β-tubulin sequences (residues 108 to 259). Selected α-tubulin and γ-tubulin sequences are included, with S. pombe α-tubulin as the designated outgroup. Parsimony analysis (100 bootstrap resamplings) was used for the consensus tree. Numbers are percentages of trees in which the group of species to the right of that branch was found. A nearly identical tree, except that E. cuniculi and S. commune branches were reversed, was obtained with a distance matrix method (500 resamplings). (Reprinted with permission from Edlind et al.,1996. )
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.fig4-5
FIGURE 5
hsp70 phylogenetic analysis, (a) Maximum parsimony (MP) bootstrap (500 replicates) consensus tree for 41 hsp70 sequences; (b) maximum likelihood (ML) consensus tree (using the PUZZLE 3.0 program with 1,000 puzzling steps) for 37 hsp70 sequences. B o t h trees demonstrate that the V. necatrix sequence is within the mitochondrial clade. Support values above branches correspond to the analysis shown. Support values below relevant branches are from analyses of t h e same data set by different methods: (a) an M L and a least squares (LS) distance (100 bootstrap replicates); (b), an M P and an LS distance analyses. Boxed values in panel b correspond to support values with the V. necatrix sequence removed from the analysis. (Reprinted with permission from Hirt et a l , 1997. )
10.1128/9781555818227/fig4-5_thmb.gif
10.1128/9781555818227/fig4-5.gif
FIGURE 5
hsp70 phylogenetic analysis, (a) Maximum parsimony (MP) bootstrap (500 replicates) consensus tree for 41 hsp70 sequences; (b) maximum likelihood (ML) consensus tree (using the PUZZLE 3.0 program with 1,000 puzzling steps) for 37 hsp70 sequences. B o t h trees demonstrate that the V. necatrix sequence is within the mitochondrial clade. Support values above branches correspond to the analysis shown. Support values below relevant branches are from analyses of t h e same data set by different methods: (a) an M L and a least squares (LS) distance (100 bootstrap replicates); (b), an M P and an LS distance analyses. Boxed values in panel b correspond to support values with the V. necatrix sequence removed from the analysis. (Reprinted with permission from Hirt et a l , 1997. )
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.fig4-6
FIGURE 6
Hypothetical phylogenetic tree.
10.1128/9781555818227/fig4-6_thmb.gif
10.1128/9781555818227/fig4-6.gif
FIGURE 6
Hypothetical phylogenetic tree.
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.fig4-7
FIGURE 7
Microsporidian phylogeny: molecular and morphologic characteristics. Bootstrap analysis (400 replicates) of t h e most parsimonious tree. Numbers represent percentages of bootstrap replicates. Characters represented are as follows. (1) Nuclear condition of the spore: D, diplokaryotic; U, uninucleate. (2) Host: C, Crustacea; I, Insecta; M, Mammalia (h, human); P, Pisces. (3) Membrane surrounding spores: N, none; P, parasitophorous vacuole; S, sporophorous vesicle. (4) Sporogony: D, disporous; O, octosporous; P, polysporous;T, tetrasporous. (5) Chromosome cycle: D l , dihaplophasic/haplophasic with meiosis; D2, dihaplophasic/haplophasic with nuclear dissociation; H, haplophasic only. Nosema sp. characters are based o n genomic placement. (Reprinted with permission from Baker et al., 1995. )
10.1128/9781555818227/fig4-7_thmb.gif
10.1128/9781555818227/fig4-7.gif
FIGURE 7
Microsporidian phylogeny: molecular and morphologic characteristics. Bootstrap analysis (400 replicates) of t h e most parsimonious tree. Numbers represent percentages of bootstrap replicates. Characters represented are as follows. (1) Nuclear condition of the spore: D, diplokaryotic; U, uninucleate. (2) Host: C, Crustacea; I, Insecta; M, Mammalia (h, human); P, Pisces. (3) Membrane surrounding spores: N, none; P, parasitophorous vacuole; S, sporophorous vesicle. (4) Sporogony: D, disporous; O, octosporous; P, polysporous;T, tetrasporous. (5) Chromosome cycle: D l , dihaplophasic/haplophasic with meiosis; D2, dihaplophasic/haplophasic with nuclear dissociation; H, haplophasic only. Nosema sp. characters are based o n genomic placement. (Reprinted with permission from Baker et al., 1995. )
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.fig4-8
FIGURE 8
Microsporidian smallsubunit rRNA phylogeny. This is a bootstrap analysis of the most parsimonious tree containing rRNA sequences from GenBank ( Table 1 ).
10.1128/9781555818227/fig4-8_thmb.gif
10.1128/9781555818227/fig4-8.gif
FIGURE 8
Microsporidian smallsubunit rRNA phylogeny. This is a bootstrap analysis of the most parsimonious tree containing rRNA sequences from GenBank ( Table 1 ).
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.fig4-9
FIGURE 9
Nosema /Vairimorpha phylogeny. This is an analysis of Nosema and Vairimorpha rRNA sequences in GenBank ( Table 1 ). As can be seen, despite the difference in nuclear number, there is overlap in these two genera at the molecular level of analysis.
10.1128/9781555818227/fig4-9_thmb.gif
10.1128/9781555818227/fig4-9.gif
FIGURE 9
Nosema /Vairimorpha phylogeny. This is an analysis of Nosema and Vairimorpha rRNA sequences in GenBank ( Table 1 ). As can be seen, despite the difference in nuclear number, there is overlap in these two genera at the molecular level of analysis.
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.fig4-10
FIGURE 10
ITS o f E. cuniculi isolates. This analysis demonstrates the variation in the number of G T TT repeats in the I TS region of t he various E. cuniculi isolates and t h e relationship to host species.
10.1128/9781555818227/fig4-10_thmb.gif
10.1128/9781555818227/fig4-10.gif
FIGURE 10
ITS o f E. cuniculi isolates. This analysis demonstrates the variation in the number of G T TT repeats in the I TS region of t he various E. cuniculi isolates and t h e relationship to host species.
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.fig4-11
FIGURE 11
PCR for E. intestinalis in stool specimens. Amplification of a band of the correct size (375 bp) is demonstrated by P C R in stool specimens from two patients with primer s e t V l : : S I 5 0 0 to the smallsubunit r R N A gene. Lanes 1 and 2, stool specimens from two patients with E. intestinalis infection; lane 3, stool from a patient with E. bieneusi infection; lane 4, negative control (no DNA).
10.1128/9781555818227/fig4-11_thmb.gif
10.1128/9781555818227/fig4-11.gif
FIGURE 11
PCR for E. intestinalis in stool specimens. Amplification of a band of the correct size (375 bp) is demonstrated by P C R in stool specimens from two patients with primer s e t V l : : S I 5 0 0 to the smallsubunit r R N A gene. Lanes 1 and 2, stool specimens from two patients with E. intestinalis infection; lane 3, stool from a patient with E. bieneusi infection; lane 4, negative control (no DNA).
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.fig4-12
FIGURE 12
In situ hybridization for E. bieneusi, demonstrating the detection of E. bieneusi in an intestinal biopsy specimen using an rRNA hybridization probe. (Reprinted with permission from Carville et al., 1997. )
10.1128/9781555818227/fig4-12_thmb.gif
10.1128/9781555818227/fig4-12.gif
FIGURE 12
In situ hybridization for E. bieneusi, demonstrating the detection of E. bieneusi in an intestinal biopsy specimen using an rRNA hybridization probe. (Reprinted with permission from Carville et al., 1997. )
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
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Tables
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia
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Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.tab4-1
TABLE 1
Microsporidian genes in GenBank a
a Adapted from Weiss and Vossbrinck (1998) with permission.
10.1128/9781555818227/tab4-1_thmb.gif
10.1128/9781555818227/tab4-1.gif
TABLE 1
Microsporidian genes in GenBank a
a Adapted from Weiss and Vossbrinck (1998) with permission.
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.tab4-2
TABLE 2
Primers for the identification and sequencing of microsporidian rDNA1 a
a Primers 18f and 1492r amplify most of the small-subunit rRNA of the microsporidia. Primers 530f and 212rl or 212r2 are used to amplify the small-subunit rRNA and the ITS region.The remaining primers are used to sequence, with overlap, the forward and reverse strands of the entire small-subunit rRNA and ITS region. Is580r amplifies a variable region of the 5' end of the large subunit rRNA gene of many microsporidia (e.g., Nosema and Vairimorpha),but it does not work on all microsporidia. ssl537 allows sequencing closer to the 3' end of the small-subunit rRNA of many but not all microsporidia. ss350f and ss350r may not be needed for sequencing reactions if 18f and 530r provide sufficient overlap to obtain clear sequence data. The table is adapted from Weiss and Vossbrinck (1998) with permission.
b ss, primers in the small-subunit rRNA gene; Is, primers in the large subunit rRNA gene; f, forward primer (positive strand); r, reverse primer (negative strand).
c Similar toV1 primer ( Zhu et al., 1993a , 1993b ; Weiss et al., 1994 ).
10.1128/9781555818227/tab4-2_thmb.gif
10.1128/9781555818227/tab4-2.gif
TABLE 2
Primers for the identification and sequencing of microsporidian rDNA1 a
a Primers 18f and 1492r amplify most of the small-subunit rRNA of the microsporidia. Primers 530f and 212rl or 212r2 are used to amplify the small-subunit rRNA and the ITS region.The remaining primers are used to sequence, with overlap, the forward and reverse strands of the entire small-subunit rRNA and ITS region. Is580r amplifies a variable region of the 5' end of the large subunit rRNA gene of many microsporidia (e.g., Nosema and Vairimorpha),but it does not work on all microsporidia. ssl537 allows sequencing closer to the 3' end of the small-subunit rRNA of many but not all microsporidia. ss350f and ss350r may not be needed for sequencing reactions if 18f and 530r provide sufficient overlap to obtain clear sequence data. The table is adapted from Weiss and Vossbrinck (1998) with permission.
b ss, primers in the small-subunit rRNA gene; Is, primers in the large subunit rRNA gene; f, forward primer (positive strand); r, reverse primer (negative strand).
c Similar toV1 primer ( Zhu et al., 1993a , 1993b ; Weiss et al., 1994 ).
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.tab4-pg146
10.1128/9781555818227/tab4-pg146_thmb.gif
10.1128/9781555818227/tab4-pg146.gif
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.tab4-pg147
10.1128/9781555818227/tab4-pg147_thmb.gif
10.1128/9781555818227/tab4-pg147.gif
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.tab4-pg148
10.1128/9781555818227/tab4-pg148_thmb.gif
10.1128/9781555818227/tab4-pg148.gif
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4
/content/10.1128/9781555818227.chap4.tab4-3
TABLE 3
Diagnostic P C R primers for microsporidia pathogenic in humans a
a Adapted from Weiss and Vossbrinck (1998) with permission.
b Designation of primers in references.
c Annealing temperature in PCR.
d Size of amplified fragment in base pairs. Eb, E. bieneusi; Ec, E. cuniculi; Ei, E. intestinalis; Eh, E. hellem.
e PstI and HaeIIIrestriction analysis differentiates these amplicons.
f HindIII and Hinfl restriction analysis differentiates these amplicons.
10.1128/9781555818227/tab4-3_thmb.gif
10.1128/9781555818227/tab4-3.gif
TABLE 3
Diagnostic P C R primers for microsporidia pathogenic in humans a
a Adapted from Weiss and Vossbrinck (1998) with permission.
b Designation of primers in references.
c Annealing temperature in PCR.
d Size of amplified fragment in base pairs. Eb, E. bieneusi; Ec, E. cuniculi; Ei, E. intestinalis; Eh, E. hellem.
e PstI and HaeIIIrestriction analysis differentiates these amplicons.
f HindIII and Hinfl restriction analysis differentiates these amplicons.
Citation:
Weiss L, Vossbrinck C.
1999.
Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia,
p 129-171.
In
Wittner M, Weiss L (ed),
The Microsporidia and Microsporidiosis.
ASM Press, Washington, DC.
doi: 10.1128/9781555818227.ch4