Synchronous detection of Burkholderia pseudomallei and its ceftazidime resistance mutation based on RNase-HII hydrolysis combined with lateral flow strip assay
ABSTRACT Melioidosis is a severe and often fatal infectious disease caused by Burkholderia pseudomallei, which poses significant challenges due to its high mortality rate and frequent misdiagnosis. To effectively treat infected patients, especially in epidemic regions like Hainan with a high resista...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-12-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.01125-23 |
| _version_ | 1797393597788585984 |
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| author | Juan Yao Zhang Zhang Shen Tian Nini Luo Jun Tan Yue Zhang Shuo Gu Qianfeng Xia |
| author_facet | Juan Yao Zhang Zhang Shen Tian Nini Luo Jun Tan Yue Zhang Shuo Gu Qianfeng Xia |
| author_sort | Juan Yao |
| collection | DOAJ |
| description | ABSTRACT Melioidosis is a severe and often fatal infectious disease caused by Burkholderia pseudomallei, which poses significant challenges due to its high mortality rate and frequent misdiagnosis. To effectively treat infected patients, especially in epidemic regions like Hainan with a high resistance rate to ceftazidime (CAZ), early diagnosis and resistance testing are critical. Here, we designed a strategy that combines RNase-HII-dependent PCR with lateral flow strip assay (LFSA) for simultaneous detection of B. pseudomallei and its CAZ resistance mutation. In this strategy, we utilized rhPCR technology to design specific primers with a C3-spacer modification. These primers target a 115-base pair region within the ORF2 of the B. pseudomallei type III secretion system gene cluster, as well as the P174L mutation in the penA gene, which is a major mutation associated with CAZ resistance in Hainan. The primers contain complementary RNA bases that can be hydrolyzed only in the presence of thermostable RNase-HII enzymes, facilitating subsequent PCR reactions. Additionally, we introduced two pairs of universal probes carrying fluorescein isothiocyanate (FITC) and biotin, as well as digoxin and biotin. The sequences of these universal probes align with specific regions of the primers, resulting in target products labeled with different tags. The resulting amplification products are then subjected to LFSA, where the presence of specific markers is visually interpreted based on color development. These markers flow to test line 1 (T1) and are captured by a fixed anti-FITC antibody, generating a red line that indicates the presence of B. pseudomallei. Similarly, the occurrence of a red line on test line 2 (T2), which is fixed with anti-digoxin antibodies, indicates the presence of the P174L mutation in B. pseudomallei. This protocol serves as an excellent tool for the rapid diagnosis of melioidosis and provides a basis for selecting appropriate antibiotic medication. It has the potential to revolutionize the diagnosis of melioidosis, particularly in resource-limited settings. Furthermore, this technique holds promise as a versatile tool for disease diagnosis in general. IMPORTANCE This study focused on the development of a reaction system using rhPCR to amplify a specific gene, ORF2, of B. pseudomallei and to identify the P174L mutation associated with increased drug resistance to ceftazidime (CAZ). The system incorporated universal primer probes and a simple temperature cycle reaction. The amplified products were then analyzed using lateral flow strip assay (LFSA) for strain identification and mutation interpretation. The developed system provides a reliable basis for diagnosing melioidosis and selecting appropriate drugs. Its potential impact is particularly significant in resource-limited settings where access to advanced diagnostic techniques is limited. This platform stands out for its simplicity, convenience, sensitivity, specificity, and portability. It shows promise as a point-of-care testing method for detecting single nucleotide polymorphism in genes associated with other diseases. By leveraging the advantages of this platform, researchers and healthcare professionals can potentially expand its use beyond melioidosis and apply it to the rapid detection of genetic variations in other disease-related genes. |
| first_indexed | 2024-03-09T00:06:08Z |
| format | Article |
| id | doaj.art-52288e9ff73d40b6af13664dc5d70191 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-03-09T00:06:08Z |
| publishDate | 2023-12-01 |
| publisher | American Society for Microbiology |
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| series | Microbiology Spectrum |
| spelling | doaj.art-52288e9ff73d40b6af13664dc5d701912023-12-12T13:17:19ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972023-12-0111610.1128/spectrum.01125-23Synchronous detection of Burkholderia pseudomallei and its ceftazidime resistance mutation based on RNase-HII hydrolysis combined with lateral flow strip assayJuan Yao0Zhang Zhang1Shen Tian2Nini Luo3Jun Tan4Yue Zhang5Shuo Gu6Qianfeng Xia7Key Laboratory of Tropical Translational Medicine of Ministry of Education, NHC Key Laboratory of Tropical Disease Control, School of Tropical Medicine, Hainan Medical University , Haikou, Hainan, ChinaKey Laboratory of Tropical Translational Medicine of Ministry of Education, NHC Key Laboratory of Tropical Disease Control, School of Tropical Medicine, Hainan Medical University , Haikou, Hainan, ChinaKey Laboratory of Tropical Translational Medicine of Ministry of Education, NHC Key Laboratory of Tropical Disease Control, School of Tropical Medicine, Hainan Medical University , Haikou, Hainan, ChinaKey Laboratory of Tropical Translational Medicine of Ministry of Education, NHC Key Laboratory of Tropical Disease Control, School of Tropical Medicine, Hainan Medical University , Haikou, Hainan, ChinaKey Laboratory of Tropical Translational Medicine of Ministry of Education, NHC Key Laboratory of Tropical Disease Control, School of Tropical Medicine, Hainan Medical University , Haikou, Hainan, ChinaKey Laboratory of Tropical Translational Medicine of Ministry of Education, NHC Key Laboratory of Tropical Disease Control, School of Tropical Medicine, Hainan Medical University , Haikou, Hainan, ChinaDepartment of Neurosurgery, Neurology Center, The First Affiliated Hospital of Hainan Medical University , Haikou, Hainan, ChinaKey Laboratory of Tropical Translational Medicine of Ministry of Education, NHC Key Laboratory of Tropical Disease Control, School of Tropical Medicine, Hainan Medical University , Haikou, Hainan, ChinaABSTRACT Melioidosis is a severe and often fatal infectious disease caused by Burkholderia pseudomallei, which poses significant challenges due to its high mortality rate and frequent misdiagnosis. To effectively treat infected patients, especially in epidemic regions like Hainan with a high resistance rate to ceftazidime (CAZ), early diagnosis and resistance testing are critical. Here, we designed a strategy that combines RNase-HII-dependent PCR with lateral flow strip assay (LFSA) for simultaneous detection of B. pseudomallei and its CAZ resistance mutation. In this strategy, we utilized rhPCR technology to design specific primers with a C3-spacer modification. These primers target a 115-base pair region within the ORF2 of the B. pseudomallei type III secretion system gene cluster, as well as the P174L mutation in the penA gene, which is a major mutation associated with CAZ resistance in Hainan. The primers contain complementary RNA bases that can be hydrolyzed only in the presence of thermostable RNase-HII enzymes, facilitating subsequent PCR reactions. Additionally, we introduced two pairs of universal probes carrying fluorescein isothiocyanate (FITC) and biotin, as well as digoxin and biotin. The sequences of these universal probes align with specific regions of the primers, resulting in target products labeled with different tags. The resulting amplification products are then subjected to LFSA, where the presence of specific markers is visually interpreted based on color development. These markers flow to test line 1 (T1) and are captured by a fixed anti-FITC antibody, generating a red line that indicates the presence of B. pseudomallei. Similarly, the occurrence of a red line on test line 2 (T2), which is fixed with anti-digoxin antibodies, indicates the presence of the P174L mutation in B. pseudomallei. This protocol serves as an excellent tool for the rapid diagnosis of melioidosis and provides a basis for selecting appropriate antibiotic medication. It has the potential to revolutionize the diagnosis of melioidosis, particularly in resource-limited settings. Furthermore, this technique holds promise as a versatile tool for disease diagnosis in general. IMPORTANCE This study focused on the development of a reaction system using rhPCR to amplify a specific gene, ORF2, of B. pseudomallei and to identify the P174L mutation associated with increased drug resistance to ceftazidime (CAZ). The system incorporated universal primer probes and a simple temperature cycle reaction. The amplified products were then analyzed using lateral flow strip assay (LFSA) for strain identification and mutation interpretation. The developed system provides a reliable basis for diagnosing melioidosis and selecting appropriate drugs. Its potential impact is particularly significant in resource-limited settings where access to advanced diagnostic techniques is limited. This platform stands out for its simplicity, convenience, sensitivity, specificity, and portability. It shows promise as a point-of-care testing method for detecting single nucleotide polymorphism in genes associated with other diseases. By leveraging the advantages of this platform, researchers and healthcare professionals can potentially expand its use beyond melioidosis and apply it to the rapid detection of genetic variations in other disease-related genes.https://journals.asm.org/doi/10.1128/spectrum.01125-23melioidosisBurkholderia pseudomalleiceftazidime resistance mutationrhPCRlateral chromatographic flow stripuniversal probes |
| spellingShingle | Juan Yao Zhang Zhang Shen Tian Nini Luo Jun Tan Yue Zhang Shuo Gu Qianfeng Xia Synchronous detection of Burkholderia pseudomallei and its ceftazidime resistance mutation based on RNase-HII hydrolysis combined with lateral flow strip assay Microbiology Spectrum melioidosis Burkholderia pseudomallei ceftazidime resistance mutation rhPCR lateral chromatographic flow strip universal probes |
| title | Synchronous detection of Burkholderia pseudomallei and its ceftazidime resistance mutation based on RNase-HII hydrolysis combined with lateral flow strip assay |
| title_full | Synchronous detection of Burkholderia pseudomallei and its ceftazidime resistance mutation based on RNase-HII hydrolysis combined with lateral flow strip assay |
| title_fullStr | Synchronous detection of Burkholderia pseudomallei and its ceftazidime resistance mutation based on RNase-HII hydrolysis combined with lateral flow strip assay |
| title_full_unstemmed | Synchronous detection of Burkholderia pseudomallei and its ceftazidime resistance mutation based on RNase-HII hydrolysis combined with lateral flow strip assay |
| title_short | Synchronous detection of Burkholderia pseudomallei and its ceftazidime resistance mutation based on RNase-HII hydrolysis combined with lateral flow strip assay |
| title_sort | synchronous detection of burkholderia pseudomallei and its ceftazidime resistance mutation based on rnase hii hydrolysis combined with lateral flow strip assay |
| topic | melioidosis Burkholderia pseudomallei ceftazidime resistance mutation rhPCR lateral chromatographic flow strip universal probes |
| url | https://journals.asm.org/doi/10.1128/spectrum.01125-23 |
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