RPA-CRISPR/Cas12a-Based Detection of <i>Haemophilus parasuis</i>
<i>Haemophilus parasuis</i> (<i>H. parasuis,</i> HPS) is a prominent pathogenic bacterium in pig production. Its infection leads to widespread fibrinous inflammation in various pig tissues and organs, often in conjunction with various respiratory virus infections, and leads t...
| Main Authors: | , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-10-01
|
| Series: | Animals |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-2615/13/21/3317 |
| _version_ | 1797632267076501504 |
|---|---|
| author | Kunli Zhang Zeyi Sun Keda Shi Dongxia Yang Zhibiao Bian Yan Li Hongchao Gou Zhiyong Jiang Nanling Yang Pinpin Chu Shaolun Zhai Zhanyong Wei Chunling Li |
| author_facet | Kunli Zhang Zeyi Sun Keda Shi Dongxia Yang Zhibiao Bian Yan Li Hongchao Gou Zhiyong Jiang Nanling Yang Pinpin Chu Shaolun Zhai Zhanyong Wei Chunling Li |
| author_sort | Kunli Zhang |
| collection | DOAJ |
| description | <i>Haemophilus parasuis</i> (<i>H. parasuis,</i> HPS) is a prominent pathogenic bacterium in pig production. Its infection leads to widespread fibrinous inflammation in various pig tissues and organs, often in conjunction with various respiratory virus infections, and leads to substantial economic losses in the pig industry. Therefore, the rapid diagnosis of this pathogen is of utmost importance. In this study, we used recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR) technology to establish a convenient detection and analysis system for <i>H. parasuis</i> that is fast to detect, easy to implement, and accurate to analyze, known as RPA-CRISPR/Cas12a analysis. The process from sample to results can be completed within 1 h with high sensitivity (0.163 pg/μL of DNA template, <i>p</i> < 0.05), which is 10<sup>4</sup> -fold higher than the common PCR method. The specificity test results show that the RPA-CRISPR/Cas12a analysis of <i>H. parasuis</i> did not react with other common pig pathogens, including <i>Streptococcus suis</i> type II and IX, <i>Actinobacillus pleuropneumoniae</i>, <i>Escherichia coli</i>, <i>Salmonella</i>, <i>Streptococcus suis</i>, and <i>Staphylococcus aureus</i> (<i>p</i> < 0.0001). The RPA-CRISPR/Cas12a assay was applied to 15 serotypes of <i>H. parasuis</i> clinical samples through crude extraction of nucleic acid by boiling method, and all of the samples were successfully identified. It greatly reduces the time and cost of nucleic acid extraction. Moreover, the method allows results to be visualized with blue light. The accurate and convenient detection method could be incorporated into a portable format as point-of-care (POC) diagnostics detection for <i>H. parasuis</i> at the field level. |
| first_indexed | 2024-03-11T11:34:35Z |
| format | Article |
| id | doaj.art-56d7524896ce4d779c1ab5f9b6240970 |
| institution | Directory Open Access Journal |
| issn | 2076-2615 |
| language | English |
| last_indexed | 2024-03-11T11:34:35Z |
| publishDate | 2023-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Animals |
| spelling | doaj.art-56d7524896ce4d779c1ab5f9b62409702023-11-10T14:57:42ZengMDPI AGAnimals2076-26152023-10-011321331710.3390/ani13213317RPA-CRISPR/Cas12a-Based Detection of <i>Haemophilus parasuis</i>Kunli Zhang0Zeyi Sun1Keda Shi2Dongxia Yang3Zhibiao Bian4Yan Li5Hongchao Gou6Zhiyong Jiang7Nanling Yang8Pinpin Chu9Shaolun Zhai10Zhanyong Wei11Chunling Li12Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Guangzhou 510640, ChinaInstitute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Guangzhou 510640, ChinaInstitute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Guangzhou 510640, ChinaInstitute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Guangzhou 510640, ChinaInstitute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Guangzhou 510640, ChinaInstitute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Guangzhou 510640, ChinaInstitute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Guangzhou 510640, ChinaInstitute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Guangzhou 510640, ChinaInstitute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Guangzhou 510640, ChinaInstitute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Guangzhou 510640, ChinaInstitute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Guangzhou 510640, ChinaCollege of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, ChinaInstitute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Guangzhou 510640, China<i>Haemophilus parasuis</i> (<i>H. parasuis,</i> HPS) is a prominent pathogenic bacterium in pig production. Its infection leads to widespread fibrinous inflammation in various pig tissues and organs, often in conjunction with various respiratory virus infections, and leads to substantial economic losses in the pig industry. Therefore, the rapid diagnosis of this pathogen is of utmost importance. In this study, we used recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR) technology to establish a convenient detection and analysis system for <i>H. parasuis</i> that is fast to detect, easy to implement, and accurate to analyze, known as RPA-CRISPR/Cas12a analysis. The process from sample to results can be completed within 1 h with high sensitivity (0.163 pg/μL of DNA template, <i>p</i> < 0.05), which is 10<sup>4</sup> -fold higher than the common PCR method. The specificity test results show that the RPA-CRISPR/Cas12a analysis of <i>H. parasuis</i> did not react with other common pig pathogens, including <i>Streptococcus suis</i> type II and IX, <i>Actinobacillus pleuropneumoniae</i>, <i>Escherichia coli</i>, <i>Salmonella</i>, <i>Streptococcus suis</i>, and <i>Staphylococcus aureus</i> (<i>p</i> < 0.0001). The RPA-CRISPR/Cas12a assay was applied to 15 serotypes of <i>H. parasuis</i> clinical samples through crude extraction of nucleic acid by boiling method, and all of the samples were successfully identified. It greatly reduces the time and cost of nucleic acid extraction. Moreover, the method allows results to be visualized with blue light. The accurate and convenient detection method could be incorporated into a portable format as point-of-care (POC) diagnostics detection for <i>H. parasuis</i> at the field level.https://www.mdpi.com/2076-2615/13/21/3317<i>Haemophilus parasuis</i>RPACRISPR/Cas12adetection |
| spellingShingle | Kunli Zhang Zeyi Sun Keda Shi Dongxia Yang Zhibiao Bian Yan Li Hongchao Gou Zhiyong Jiang Nanling Yang Pinpin Chu Shaolun Zhai Zhanyong Wei Chunling Li RPA-CRISPR/Cas12a-Based Detection of <i>Haemophilus parasuis</i> Animals <i>Haemophilus parasuis</i> RPA CRISPR/Cas12a detection |
| title | RPA-CRISPR/Cas12a-Based Detection of <i>Haemophilus parasuis</i> |
| title_full | RPA-CRISPR/Cas12a-Based Detection of <i>Haemophilus parasuis</i> |
| title_fullStr | RPA-CRISPR/Cas12a-Based Detection of <i>Haemophilus parasuis</i> |
| title_full_unstemmed | RPA-CRISPR/Cas12a-Based Detection of <i>Haemophilus parasuis</i> |
| title_short | RPA-CRISPR/Cas12a-Based Detection of <i>Haemophilus parasuis</i> |
| title_sort | rpa crispr cas12a based detection of i haemophilus parasuis i |
| topic | <i>Haemophilus parasuis</i> RPA CRISPR/Cas12a detection |
| url | https://www.mdpi.com/2076-2615/13/21/3317 |
| work_keys_str_mv | AT kunlizhang rpacrisprcas12abaseddetectionofihaemophilusparasuisi AT zeyisun rpacrisprcas12abaseddetectionofihaemophilusparasuisi AT kedashi rpacrisprcas12abaseddetectionofihaemophilusparasuisi AT dongxiayang rpacrisprcas12abaseddetectionofihaemophilusparasuisi AT zhibiaobian rpacrisprcas12abaseddetectionofihaemophilusparasuisi AT yanli rpacrisprcas12abaseddetectionofihaemophilusparasuisi AT hongchaogou rpacrisprcas12abaseddetectionofihaemophilusparasuisi AT zhiyongjiang rpacrisprcas12abaseddetectionofihaemophilusparasuisi AT nanlingyang rpacrisprcas12abaseddetectionofihaemophilusparasuisi AT pinpinchu rpacrisprcas12abaseddetectionofihaemophilusparasuisi AT shaolunzhai rpacrisprcas12abaseddetectionofihaemophilusparasuisi AT zhanyongwei rpacrisprcas12abaseddetectionofihaemophilusparasuisi AT chunlingli rpacrisprcas12abaseddetectionofihaemophilusparasuisi |