Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples
Abstract
:1. Introduction
2. Results
3. Discussion
4. Materials and Methods
4.1. Sample Materials
4.2. Nucleic Acid Extraction and Internal Amplification Control
4.3. Applied Target-Specific PCRs
4.4. Applied Non-Target-Specific PCR
4.5. Statistical Assessment
4.6. Ethical Clearance
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Real-Time PCR Assay | GenBank Accession Number | Sequence |
PCR 1 | AF023245 | 5′- AACACGGACCCACCAGGTTGATTCTGCCTGACGTAGATGCTAGTCTCTGAGATTAAGCCATGCATGTCAGTGAAGCCTTACGGTGGAACGGCGAACGGCTCAGTAATGTTGCGGTAATTTGGTCTCTGTGTGTAAACTAACCACGGTAACCTGTGGCTAAAAGCGGAGAATAAGGCGCAACCCTATCAGCTTGTTGGTAGTGTAAAGGACTACCAAGGCCATGACGGGTAACGGGAAATCAGGGTTTGATTCCGGAGAGGGAGCCTGAGAGATGGCTCCCACGTCCAAGGACGGCAGCAGGCGCGAAACTTGTCCACTCCTTACGGGGGAGACAGTCATGAGACGTGAGTATAAGACCTGAGTGTAAAGACCTTAGGGTGAAGCAATTGGAGGGCAAGCTTTGGTGCCAGCAGC-3′ |
PCR 2 | KC513629.1 | 5′- GACGTAGTAGCCATCTCTCAGGCTCCCTCTCCGGAACCAAACCCTGATCCCCCGTATCCCGTCTGCGCCTAGTTAGGCCATTACCCTAACTACCAGCTGATAGGCCCACAACTTACTTGCCAACCCCCACAGGGGCAGACCACTATCTGCAGTTTCCCGCAGCTACTGCTCATCCCGCAAACAAATCATCGTGCTATCACTGAGCCGTCCGCTAATCCCCCACAAAGAGTTCACAAGCATGCATGGCTTAGCCCCAGAGAATAGCATCCACGTCAGGCAGAATCAACCTGATGCCCCACA-3′ |
PCR 3 | AF024657 | 5′- GGAAAACTTACCAGGGTCAAGTCATTCGTTGATCGAATACGTGAGAATGGCAGGAGTGGTGCATGGCCGTTGGAAATTGATGGGGCGACCTTTAGCTTAAATGCTTAAACCAGTGAGACCTCCTTGACAGGTGTTCTGTAACACAGGAGGGTGGAGGCTATAACAGGTCCGTGATGCCCTTAGATATCCTGGGCAGCAAGCGCAATACAATATCTCTTCAGT-3′ |
PCR 4 | AF023245 | 5′- GGTGCGGTGGTGTGTGCAGGCGTGAGAGTGTATCTGCAAGTGTGAGGGATGTGGGTGCAGCGAGTTAGAGGTGGTTCCATGTGGAATAGTGGGATTGGTACGTGATGGTTGGATGGGGGAATGAT-3′ |
PCR 5 | U09929 | 5′- AGGATCATAACACCAGGTTGATTCTGCCTGACGTGGATGCTATTCTCTGGGACTAAGCCATGCATGTTGATGAACCTTGTGGGGGATTGACGGACGGCTCAGTGATAGTACGATGATTTGGTTGGCGGGAGAGCTGTAACTGCGGGAAACTGCAGGTAGGGGGCTAGGAGTGTTTTTGACACGAGCCAAGTAAGTTGTAGGCCTATCAGCTGGTAGTTAGGGTAATGGCCTAACTAGGCGGAGACGG-3′ |
PCR 6 | GU291265 | 5′- GAACCTGCGGAAGGATCATTAACGCGCAAGAGGTCGAAGTTGGCCCCCGAAGCTCTTCCGTCTCCCCCCCGGGCCTCCCGGGGAGGTTGCGGGCGGCGAGGGGTGCC-3′ |
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Assay or Assay Combination | n | Positives (%) | CT Value Mean (SD), Median (Min, Max)a | Sensitivity (0.95 CI) | Specificity (0.95 CI) |
---|---|---|---|---|---|
PCR 1 | 1339 | 87 (6.50) | 24.98 (5.32), 25.70 (14.30, 34.10) | 0.974 (0.899, 0.994) | 0.991 (0.984, 0.995) |
PCR 2 | 1339 | 47 (3.51) | 29.84 (3.59), 30.10 (23.20, 35.80) | 0.604 (0.492, 0.707) | 1 (n.e.) |
PCR 3 + 5 | 1339 | 77 (5.75) | 30.22 (5.12), 30.60 (20.50, 38.30) | 0.950 (0.868, 0.982) | 0.998 (0.992, 0.999) |
PCR 4 + 5 | 1339 | 84 (6.27) | 30.79 (5.34), 31.40 (19.90, 39.50) | 0.961 (0.884, 0.988) | 0.993 (0.986, 0.996) |
Kappa (0.95 CI) | 1339 | 0.796 (0.742, 0.834) |
PCR 1 | PCR 2 | PCR 3 | PCR 4 | PCR 5 | PCR 6 | |
---|---|---|---|---|---|---|
Target specificity | Small subunit ribosomal RNA gene of Enterocytozoon bieneusi, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis | Small subunit ribosomal RNA gene of Enterocytozoon bieneusi, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis | Small subunit ribosomal RNA gene of Enterocytozoon bieneusi | Internal transcribed spacer (ITS) sequence of Enterocytozoon bieneusi | Small subunit ribosomal RNA gene of Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis | Internal transcribed spacer (ITS) sequence of the non-target microorganism Microsporidium spp. |
Amplicon length | 394 base pairs | 280 base pairs | 202 base pairs | 105 base pairs | 227 base pairs | 87 base pairs |
Cycle number | 50 | 40 | 40 | 50 | 40 | 45 |
Forward primer 1 | 5′-CACCAGGTTGATTCTGCCTGA-3′ | 5′-CAGGTTGATTCTGCCTGACG-3′ | 5′-CCAGGGTCAAGTCATTCGTT-3′ | 5′-TGTGTAGGCGTGAGAGTGTATCTG-3′ | 5′-CACCAGGTTGATTCTGCCTGAC-3′ | 5′-TCTTGCGCGTTAATGATCCTT-3′ |
Forward primer 2 | 5′-TCCGGAGAGGGAGCCTGAG-3′ | n.a. | n.a. | n.a. | n.a. | n.a. |
Reverse primer 1 | 5′-GCTTGCCCTCCAATTGCTTC-3′ | 5′-CCATCTCTCAGGCTCCCTC-3′ | 5′-TATTGTATTGCGCTTGCTGC-3′ | 5′-CATCCAACCATCACGTACCAATC-3′ | 5′-CTAGTTAGGCCATTACCCTAACTACCA-3′ | 5′-AGGTTGCGGGCGGC-3′ |
Reverse primer 2 | 5′-GACTTGCCCTCCAATCACATG-3′ | n.a. | n.a. | n.a. | n.a. | n.a. |
Reverse primer 3 | 5′-CCGACTTGCCCTCCAGTAAA-3′ | n.a. | n.a. | n.a. | n.a. | n.a. |
Reverse primer 4 | 5′-CTTGGCCTCCAATCAATCTCG-3′ | n.a. | n.a. | n.a. | n.a. | n.a. |
Hybridization probe * | 5′-TGGCAGCAGGCGCGAAACTTGT-3′ | n.a. | 5′-GATGCCCTTAGATATCCTGG-3′ | 5′-CACTGCACCCACATCCCTCACCCTT-3′ | 5′-CTATCACTGAG+C+CGT+CC-3′ | 5′-ACGGAAGAGCTTCGGGGGCCA-3′ |
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Tanida, K.; Hahn, A.; Eberhardt, K.A.; Tannich, E.; Landt, O.; Kann, S.; Feldt, T.; Sarfo, F.S.; Di Cristanziano, V.; Frickmann, H.; Loderstädt, U. Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples. Pathogens 2021, 10, 656. https://doi.org/10.3390/pathogens10060656
Tanida K, Hahn A, Eberhardt KA, Tannich E, Landt O, Kann S, Feldt T, Sarfo FS, Di Cristanziano V, Frickmann H, Loderstädt U. Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples. Pathogens. 2021; 10(6):656. https://doi.org/10.3390/pathogens10060656
Chicago/Turabian StyleTanida, Konstantin, Andreas Hahn, Kirsten Alexandra Eberhardt, Egbert Tannich, Olfert Landt, Simone Kann, Torsten Feldt, Fred Stephen Sarfo, Veronica Di Cristanziano, Hagen Frickmann, and Ulrike Loderstädt. 2021. "Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples" Pathogens 10, no. 6: 656. https://doi.org/10.3390/pathogens10060656