Development of taqman-based real-time rt-pcr assay for quantitative detection of feline morbillivirus (feMV) and phylogenetic analysis of Malaysian feMV isolates
Feline morbillivirus (FeMV) of genus Morbillivirus is a novel emerging virus of domestic cats, linked with the development of CKD. Several quantitative diagnostic assays have been developed for the detection of FeMV such as reverse transcriptase loop-mediated isothermal amplification (RT-LAMP)...
| Main Author: | |
|---|---|
| Format: | Thesis |
| Language: | English |
| Published: |
2021
|
| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/97870/1/FPV%202021%202%20UPMIR.pdf |
| _version_ | 1796983491787751424 |
|---|---|
| author | Makhtar, Siti Tasnim |
| author_facet | Makhtar, Siti Tasnim |
| author_sort | Makhtar, Siti Tasnim |
| collection | UPM |
| description | Feline morbillivirus (FeMV) of genus Morbillivirus is a novel emerging virus of
domestic cats, linked with the development of CKD. Several quantitative
diagnostic assays have been developed for the detection of FeMV such as reverse
transcriptase loop-mediated isothermal amplification (RT-LAMP) and
quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR);
however, none of the published assays targets the N gene of FeMV. N gene is one
of the most conserved genes in morbilliviruses which also plays an important role
in RNA synthesis by RdRp complex. In view for a specific and sensitive assay
targeting local isolates, the objectives of this study were to develop Taqman-based
qRT-PCR assay for the quantitative measurement of FeMV based on the sequence
of N gene of local isolates and to assess the sensitivity and specificity of the
developed qPCR assay in detecting FeMV. Sequence analyses of FeMV-Malaysia
isolates were performed to develop specific primers targeting N gene.
Phylogenetic analysis shown that the local isolates were closely related to the
isolates from China, Thailand and Japan. Subsequently, a set of primers was
designed and used in qRT-PCR assay which demonstrated a high specificity with
no amplification signal towards other morbilliviruses and other feline viruses.
Lowest limit of detection for the developed assay was at 1.74 x 10-4 copies/ߤL.
The CV values for inter- and intra-assay variation were low, ranging from 1.38% -
2.03%, and 0.34% - 0.53%, respectively. Besides, the developed qRT-PCR assay
was tested using cats’ urine and kidney samples. The findings were then
compared with the detections using conventional RT-PCR. The developed qRT-PCR assay detected FeMV in 35.2% of cats’ samples compared to 15.5% by
conventional RT-PCR. In conclusion, the developed assay of qRT-PCR has a high
specificity and sensitivity in detecting FeMV compared to conventional RT-PCR,
thus can be utilized as diagnostic tools and to determine possible association with
CKD occurrence in cats. |
| first_indexed | 2024-03-06T11:07:05Z |
| format | Thesis |
| id | upm.eprints-97870 |
| institution | Universiti Putra Malaysia |
| language | English |
| last_indexed | 2024-03-06T11:07:05Z |
| publishDate | 2021 |
| record_format | dspace |
| spelling | upm.eprints-978702022-07-05T08:47:30Z http://psasir.upm.edu.my/id/eprint/97870/ Development of taqman-based real-time rt-pcr assay for quantitative detection of feline morbillivirus (feMV) and phylogenetic analysis of Malaysian feMV isolates Makhtar, Siti Tasnim Feline morbillivirus (FeMV) of genus Morbillivirus is a novel emerging virus of domestic cats, linked with the development of CKD. Several quantitative diagnostic assays have been developed for the detection of FeMV such as reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR); however, none of the published assays targets the N gene of FeMV. N gene is one of the most conserved genes in morbilliviruses which also plays an important role in RNA synthesis by RdRp complex. In view for a specific and sensitive assay targeting local isolates, the objectives of this study were to develop Taqman-based qRT-PCR assay for the quantitative measurement of FeMV based on the sequence of N gene of local isolates and to assess the sensitivity and specificity of the developed qPCR assay in detecting FeMV. Sequence analyses of FeMV-Malaysia isolates were performed to develop specific primers targeting N gene. Phylogenetic analysis shown that the local isolates were closely related to the isolates from China, Thailand and Japan. Subsequently, a set of primers was designed and used in qRT-PCR assay which demonstrated a high specificity with no amplification signal towards other morbilliviruses and other feline viruses. Lowest limit of detection for the developed assay was at 1.74 x 10-4 copies/ߤL. The CV values for inter- and intra-assay variation were low, ranging from 1.38% - 2.03%, and 0.34% - 0.53%, respectively. Besides, the developed qRT-PCR assay was tested using cats’ urine and kidney samples. The findings were then compared with the detections using conventional RT-PCR. The developed qRT-PCR assay detected FeMV in 35.2% of cats’ samples compared to 15.5% by conventional RT-PCR. In conclusion, the developed assay of qRT-PCR has a high specificity and sensitivity in detecting FeMV compared to conventional RT-PCR, thus can be utilized as diagnostic tools and to determine possible association with CKD occurrence in cats. 2021-04 Thesis NonPeerReviewed text en http://psasir.upm.edu.my/id/eprint/97870/1/FPV%202021%202%20UPMIR.pdf Makhtar, Siti Tasnim (2021) Development of taqman-based real-time rt-pcr assay for quantitative detection of feline morbillivirus (feMV) and phylogenetic analysis of Malaysian feMV isolates. Masters thesis, Universiti Putra Malaysia. Veterinary virology Morbilliviruses Cats - Viruses |
| spellingShingle | Veterinary virology Morbilliviruses Cats - Viruses Makhtar, Siti Tasnim Development of taqman-based real-time rt-pcr assay for quantitative detection of feline morbillivirus (feMV) and phylogenetic analysis of Malaysian feMV isolates |
| title | Development of taqman-based real-time rt-pcr assay for quantitative detection of feline morbillivirus (feMV) and phylogenetic analysis of Malaysian feMV isolates |
| title_full | Development of taqman-based real-time rt-pcr assay for quantitative detection of feline morbillivirus (feMV) and phylogenetic analysis of Malaysian feMV isolates |
| title_fullStr | Development of taqman-based real-time rt-pcr assay for quantitative detection of feline morbillivirus (feMV) and phylogenetic analysis of Malaysian feMV isolates |
| title_full_unstemmed | Development of taqman-based real-time rt-pcr assay for quantitative detection of feline morbillivirus (feMV) and phylogenetic analysis of Malaysian feMV isolates |
| title_short | Development of taqman-based real-time rt-pcr assay for quantitative detection of feline morbillivirus (feMV) and phylogenetic analysis of Malaysian feMV isolates |
| title_sort | development of taqman based real time rt pcr assay for quantitative detection of feline morbillivirus femv and phylogenetic analysis of malaysian femv isolates |
| topic | Veterinary virology Morbilliviruses Cats - Viruses |
| url | http://psasir.upm.edu.my/id/eprint/97870/1/FPV%202021%202%20UPMIR.pdf |
| work_keys_str_mv | AT makhtarsititasnim developmentoftaqmanbasedrealtimertpcrassayforquantitativedetectionoffelinemorbillivirusfemvandphylogeneticanalysisofmalaysianfemvisolates |