A Thermostabilized, One-Step PCR Assay for Simultaneous Detection of Klebsiella pneumoniae and Haemophilus influenzae
Klebsiella pneumoniae and Haemophilus influenzae are two common pathogens associated with respiratory tract infections. The identification of these pathogens using conventional molecular diagnostic tests requires trained personnel, cold-chain transportation, and storage-dependance, which does not...
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| Format: | Article |
| Language: | English |
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Hindawi Publishing Corporation
2017
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| Online Access: | http://eprints.usm.my/38218/1/A_Thermostabilized%2C_One-Step_PCR_Assay_for.pdf |
| _version_ | 1825833809816322048 |
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| author | Khazani, Nur Amalina Nik Mohd Noor, Nik Zuraina Chan, Yean Yean Hasan, Habsah Md Noor, Siti Suraiya Mohamad, Suharni |
| author_facet | Khazani, Nur Amalina Nik Mohd Noor, Nik Zuraina Chan, Yean Yean Hasan, Habsah Md Noor, Siti Suraiya Mohamad, Suharni |
| author_sort | Khazani, Nur Amalina |
| collection | USM |
| description | Klebsiella pneumoniae and Haemophilus influenzae are two common pathogens associated with respiratory tract infections.
The identification of these pathogens using conventional molecular diagnostic tests requires trained personnel, cold-chain
transportation, and storage-dependance, which does not render them user-friendly. The aim of this study was to develop a
thermostabilized, cold-chain-free, one-step multiplex PCR for simultaneous detection of K. pneumoniae and H. influenzae. The
multiplex PCR assay was designed to amplify the php gene of K. pneumoniae (202 bp) and p6 gene of H. influenzae (582 bp).
In addition, the specific primer to amplify glm gene of Helicobacter pylori (105 bp) was included as an internal amplification
control. Subsequently, the designed primers and all PCR reagents were thermostabilized by lyophilization. The stability of the
thermostabilized PCR was evaluated using the Q10 method. The sensitivity and specificity of performances for thermostabilized
PCR were evaluated using 127 clinical isolates and were found to be 100% sensitive and specific.The thermostabilized PCR mix was
found to be stable for 30 days and the Q10 accelerated stability was found to be 3.02 months. A cold-chain-free, PCR assay for easy,
rapid, and simultaneous detection of K. pneumoniae and H. influenzae was successfully developed in this study. |
| first_indexed | 2024-03-06T15:13:22Z |
| format | Article |
| id | usm.eprints-38218 |
| institution | Universiti Sains Malaysia |
| language | English |
| last_indexed | 2024-03-06T15:13:22Z |
| publishDate | 2017 |
| publisher | Hindawi Publishing Corporation |
| record_format | dspace |
| spelling | usm.eprints-382182018-01-04T04:54:08Z http://eprints.usm.my/38218/ A Thermostabilized, One-Step PCR Assay for Simultaneous Detection of Klebsiella pneumoniae and Haemophilus influenzae Khazani, Nur Amalina Nik Mohd Noor, Nik Zuraina Chan, Yean Yean Hasan, Habsah Md Noor, Siti Suraiya Mohamad, Suharni QR1-502 Microbiology RK1-715 Dentistry Klebsiella pneumoniae and Haemophilus influenzae are two common pathogens associated with respiratory tract infections. The identification of these pathogens using conventional molecular diagnostic tests requires trained personnel, cold-chain transportation, and storage-dependance, which does not render them user-friendly. The aim of this study was to develop a thermostabilized, cold-chain-free, one-step multiplex PCR for simultaneous detection of K. pneumoniae and H. influenzae. The multiplex PCR assay was designed to amplify the php gene of K. pneumoniae (202 bp) and p6 gene of H. influenzae (582 bp). In addition, the specific primer to amplify glm gene of Helicobacter pylori (105 bp) was included as an internal amplification control. Subsequently, the designed primers and all PCR reagents were thermostabilized by lyophilization. The stability of the thermostabilized PCR was evaluated using the Q10 method. The sensitivity and specificity of performances for thermostabilized PCR were evaluated using 127 clinical isolates and were found to be 100% sensitive and specific.The thermostabilized PCR mix was found to be stable for 30 days and the Q10 accelerated stability was found to be 3.02 months. A cold-chain-free, PCR assay for easy, rapid, and simultaneous detection of K. pneumoniae and H. influenzae was successfully developed in this study. Hindawi Publishing Corporation 2017 Article PeerReviewed application/pdf en http://eprints.usm.my/38218/1/A_Thermostabilized%2C_One-Step_PCR_Assay_for.pdf Khazani, Nur Amalina and Nik Mohd Noor, Nik Zuraina and Chan, Yean Yean and Hasan, Habsah and Md Noor, Siti Suraiya and Mohamad, Suharni (2017) A Thermostabilized, One-Step PCR Assay for Simultaneous Detection of Klebsiella pneumoniae and Haemophilus influenzae. Journal of Tropical Medicine, 2017 (721084). pp. 1-8. ISSN 1687-9686 https://doi.org/10.1155/2017/7210849 |
| spellingShingle | QR1-502 Microbiology RK1-715 Dentistry Khazani, Nur Amalina Nik Mohd Noor, Nik Zuraina Chan, Yean Yean Hasan, Habsah Md Noor, Siti Suraiya Mohamad, Suharni A Thermostabilized, One-Step PCR Assay for Simultaneous Detection of Klebsiella pneumoniae and Haemophilus influenzae |
| title | A Thermostabilized, One-Step PCR Assay for Simultaneous Detection of Klebsiella pneumoniae and Haemophilus influenzae |
| title_full | A Thermostabilized, One-Step PCR Assay for Simultaneous Detection of Klebsiella pneumoniae and Haemophilus influenzae |
| title_fullStr | A Thermostabilized, One-Step PCR Assay for Simultaneous Detection of Klebsiella pneumoniae and Haemophilus influenzae |
| title_full_unstemmed | A Thermostabilized, One-Step PCR Assay for Simultaneous Detection of Klebsiella pneumoniae and Haemophilus influenzae |
| title_short | A Thermostabilized, One-Step PCR Assay for Simultaneous Detection of Klebsiella pneumoniae and Haemophilus influenzae |
| title_sort | thermostabilized one step pcr assay for simultaneous detection of klebsiella pneumoniae and haemophilus influenzae |
| topic | QR1-502 Microbiology RK1-715 Dentistry |
| url | http://eprints.usm.my/38218/1/A_Thermostabilized%2C_One-Step_PCR_Assay_for.pdf |
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