Bioscience Method 2024, Vol.15 http://bioscipublisher.com/index.php/bm © 2024 BioSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved.
Bioscience Method 2024, Vol.15 http://bioscipublisher.com/index.php/bm © 2024 BioSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. BioSci Publisher is an international Open Access publisher specializing in bioscience methods, including technology, lab tool, statistical software and relative fields registered at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada. Publisher BioSci Publisher Editedby Editorial Team of Bioscience Methods Email: edit@bm.bioscipublisher.com Website: http://bioscipublisher.com/index.php/bm Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada Bioscience Methods (ISSN 1925-1920) is an open access, peer reviewed journal published online by BioSci Publisher. The journal publishes all the latest and outstanding research articles, letters and reviews in all areas of bioscience, the range of topics including (but are not limited to) technology review, technique know-how, lab tool, statistical software and known technology modification. Case studies on technologies for gene discovery and function validation as well as genetic transformation. All the articles published in Bioscience Methods are Open Access, and are distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BioSci Publisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights.
Bioscience Methods (online), 2024, Vol.15, No.3 ISSN 1925-1920 https://bioscipublisher.com/index.php/bm © 2024 BioSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. Latest Content Molecular Diagnostics: A New Era in Pet Disease Detection Xinghao Li, Jia Xuan Bioscience Methods, 2024, Vol.15, No.3, 91-101 Transcriptomic Approaches to Studying Rice Pathogen Interactions Yumin Huang Bioscience Methods, 2024, Vol.15, No.3, 102-113 Metabolic Engineering of Tea: Enhancing Bioactive Compound Production Chunyu Li, Baofu Huang Bioscience Methods, 2024, Vol.15, No.3, 114-123 Development of AI-Based Diagnostic Systems for Hypertensive Heart Disease Jianli Zhong Bioscience Methods, 2024, Vol.15, No.3, 124-138 Advances in Biotechnological Approaches to Enhance Insect Resistance in Sugarcane Kaiwen Liang Bioscience Methods, 2024, Vol.15, No.3, 139-148
Bioscience Methods 2024, Vol.15, No.3, 91-101 http://bioscipublisher.com/index.php/bm 91 Review Article Open Access Molecular Diagnostics: A New Era in Pet Disease Detection Xinghao Li, Jia Xuan Institute of Life Science, Jiyang College of Zhejiang A&F University, Zhuji, 311800, Zhejiang, China Corresponding email: jia.xuan@jicat.org Bioscience Methods, 2024, Vol.15, No.3 doi: 10.5376/bm.2024.15.0011 Received: 26 Mar., 2024 Accepted: 21 May, 2024 Published: 31 May, 2024 Copyright © 2024 Li and Xuan, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Li X.H., and Xuan J., 2024, Molecular diagnostics: a new era in pet disease detection, Bioscience Methods, 15(3): 91-101 (doi: 10.5376/bm.2024.15.0011) Abstract The field of pet disease detection has traditionally relied on conventional diagnostic methods, which, while effective, often lack the sensitivity and specificity required for early and accurate disease identification. The emergence of molecular diagnostics has revolutionized this landscape, offering precise, rapid, and comprehensive tools for detecting a wide range of diseases in pets. This study explores the fundamentals of molecular diagnostics, including key concepts and techniques such as Polymerase Chain Reaction (PCR), Next-Generation Sequencing (NGS), microarrays, and CRISPR-based diagnostics, highlighting their advantages over traditional methods. We examine the applications of these technologies in the detection of infectious diseases, genetic disorders, oncology, and chronic disease management in pets. The study also delves into recent technological advancements, including improvements in PCR technology, innovations in sequencing platforms, the integration of artificial intelligence, and the development of portable diagnostic tools. Despite the significant promise of molecular diagnostics, challenges such as technical limitations, cost, accessibility, ethical concerns, and regulatory issues remain. A detailed case study illustrates the practical application of these diagnostics in a real-world veterinary scenario, offering insights into outcomes and future directions. Finally, we discuss the future potential of molecular diagnostics in personalized veterinary medicine, its integration with telemedicine, and its role in preventive care. This study underscores the transformative impact of molecular diagnostics on veterinary practice and calls for further research to overcome existing challenges and fully realize its potential. Keywords Molecular diagnostics; Pet disease detection; Veterinary oncology; Genetic screening; Preventive veterinary medicine 1 Introduction Traditionally, the detection of diseases in pets has relied heavily on clinical signs, physical examinations, and a variety of laboratory tests such as blood work, urinalysis, and imaging techniques like X-rays and ultrasounds. These methods, while useful, often lack the sensitivity and specificity required for early and accurate diagnosis. For instance, imaging techniques like CT and MRI primarily detect anatomical changes, which may not appear until the disease has progressed significantly (Phelps, 2000). Additionally, traditional diagnostic methods can be time-consuming and may not always provide a definitive diagnosis, leading to delays in treatment and potentially poorer outcomes for the pet. The advent of molecular diagnostics has revolutionized the field of veterinary medicine by providing tools that can detect diseases at a molecular level, often before clinical signs become apparent. Techniques such as polymerase chain reaction (PCR), next-generation sequencing, and positron emission tomography (PET) have significantly improved the sensitivity and specificity of disease detection (Daniels, 2013; Granberg et al., 2014). Molecular diagnostics allow for the identification of pathogens, genetic mutations, and other molecular markers associated with disease, enabling earlier and more accurate diagnosis. For example, PET imaging can detect metabolic abnormalities in diseases like cancer and neurological disorders long before anatomical changes are visible (Phelps, 2000; Sala and Perani, 2019). Early and accurate diagnosis is crucial in veterinary medicine as it can significantly improve the prognosis and quality of life for pets. Early detection allows for timely intervention, which can prevent the progression of the disease and reduce the severity of symptoms. Accurate diagnosis ensures that the appropriate treatment is administered, thereby increasing the chances of successful outcomes. Molecular diagnostics play a vital role in this regard by providing precise and early detection of diseases, which is particularly important for conditions that are difficult to diagnose using traditional methods (Daniels, 2013; Arora et al., 2023). For instance, the use of
Bioscience Methods 2024, Vol.15, No.3, 91-101 http://bioscipublisher.com/index.php/bm 92 molecular diagnostics in detecting infectious diseases in pets has led to better management and control of these diseases, ultimately improving animal health and welfare (Balamurugan et al., 2010; Granberg et al., 2014). This study explores the advancements in molecular diagnostics and their impact on pet disease detection; provides a comprehensive overview of the current state of molecular diagnostics in veterinary medicine, and highlights the benefits and challenges associated with these techniques. By examining various molecular diagnostic methods and their applications, this study seeks to underscore the importance of early and accurate diagnosis in improving the health outcomes of pets. The scope includes an analysis of different molecular diagnostic tools, their clinical applications, and the future prospects of molecular diagnostics in veterinary medicine. 2 Fundamentals of Molecular Diagnostics 2.1 Definition and key concepts Molecular diagnostics refers to a collection of techniques used to analyze biological markers in the genome and proteome. These techniques are employed to diagnose and monitor diseases, detect risk, and decide which therapies will work best for individual patients. The key concepts include the detection and quantification of nucleic acids (DNA and RNA), proteins, and other molecules that provide information about the health status of an organism. 2.2 Types of molecular diagnostic techniques Polymerase Chain Reaction (PCR) is a widely used method in molecular diagnostics due to its high sensitivity and specificity. PCR amplifies small segments of DNA or RNA, making it possible to detect even minute quantities of pathogens or genetic mutations. This technique has been extensively applied in various fields, including dentistry, where it helps in the detection and identification of microorganisms responsible for periodontal and endodontic infections (Shahi et al., 2018). PCR's ability to provide rapid and accurate results makes it a cornerstone in the diagnosis of infectious diseases (Kurkela and Brown, 2009). Next-Generation Sequencing (NGS) is a powerful tool that allows for the comprehensive analysis of genetic material. NGS can sequence entire genomes or specific areas of interest, providing detailed information about genetic variations and mutations. This technology has revolutionized the field of molecular diagnostics by enabling the identification of pathogens and genetic disorders with high precision. NGS is particularly useful in the investigation of fevers of unknown origin (FUO) and has the potential to replace traditional microbial identification methods (Wright et al., 2021). Microarrays are another important molecular diagnostic technique that allows for the simultaneous analysis of thousands of genetic sequences. This method is highly sensitive and can detect multiple pathogens in a single test. However, the high cost and complexity of microarrays limit their routine use in clinical diagnostics. Despite these limitations, microarrays hold promise for the future of molecular diagnostics, especially in the detection of genetic polymorphisms and immune markers (Kurkela and Brown, 2009). CRISPR-based diagnostics represent a cutting-edge approach in molecular diagnostics. Leveraging the CRISPR-Cas system, these techniques offer high sensitivity and specificity for nucleic acid detection. CRISPR diagnostics are particularly advantageous for point-of-care testing due to their simplicity and rapid turnaround time. They have been successfully used to detect various pathogens, including SARS-CoV-2, and have shown potential for use in low-resource settings (Figure 1) (Hatoum-Aslan, 2018; Kaminski et al., 2021; Abudayyeh and Gootenberg, 2021). The development of CRISPR-based tools continues to expand, providing new opportunities for accurate and accessible disease detection (Yin et al., 2020; Weng et al., 2023). 2.3 Advantages over traditional methods Molecular diagnostic techniques offer several advantages over traditional methods. They provide higher sensitivity and specificity, allowing for the detection of low levels of pathogens or genetic mutations. These techniques also offer faster turnaround times, which is crucial for timely diagnosis and treatment. Additionally, molecular diagnostics can be performed on a variety of sample types, including blood, tissue, and swabs, making them versatile tools in disease detection. The ability to perform multiplex testing, where multiple
Bioscience Methods 2024, Vol.15, No.3, 91-101 http://bioscipublisher.com/index.php/bm 93 targets are analyzed simultaneously, further enhances the efficiency and effectiveness of molecular diagnostics (Kurkela and Brown, 2009; Cai et al., 2014; Wright et al., 2021). Figure 1 Multiplexed CRISPR-based target detection (Adopted from Kaminski et al., 2021) Image caption: Left: pooled multiplexing of up to four targets, as implemented in SHERLOCKv2. Orthogonal CRISPR enzymes (PsmCas13b, LwaCas13a, CcaCas13b, AsCas12a), each cleaving preferentially different reporter molecules bearing different fluorophores (FAM, TEX615 (TEX), Cy5, hexachlorofluorescein (HEX)) with distinct absorbance and emission wavelengths. Right: in CARMEN, a distinct colour code is added to each PCR-amplified sample or Cas13-based detection mix. The Cas13-based detection mix contains Cas13a, a sequence-specific crRNA and a cleavage reporter. The colour-coded solutions are emulsified in fluorous oil, which creates nanolitre droplets. Droplets from all samples and detection mixes are pooled together and loaded into a microwell-array chip in one step to create all possible pairwise combinations. The droplet pair in each well is identified using fluorescence microscopy before they are merged by exposure to an electric field. Fluorescence microscopy is then used to monitor each Cas13-based detection reaction (Adopted from Kaminski et al., 2021) 3 Applications in Pet Disease Detection 3.1 Infectious disease detection Molecular diagnostics have revolutionized the detection of viral infections in pets. Techniques such as polymerase chain reaction (PCR) and droplet digital PCR (ddPCR) are highly sensitive and can detect viral DNA or RNA even in low quantities. These methods are particularly useful for identifying viral pathogens in both symptomatic and asymptomatic animals, aiding in early diagnosis and management (Lappin et al., 2009; Arora et al., 2023). The identification of bacterial infections in pets has also benefited from molecular diagnostics. PCR-based assays can detect bacterial DNA in various samples, including blood, tissue, and bodily fluids. This allows for rapid and accurate diagnosis, which is crucial for the timely treatment of bacterial infections. Molecular techniques are employed to detect parasitic infections in pets. These methods can identify the genetic material of parasites, providing a precise diagnosis that is essential for effective treatment. The use of molecular diagnostics in parasitology helps in detecting infections that might be missed by traditional methods. 3.2 Genetic disease screening Molecular diagnostics play a significant role in screening for inherited diseases in dogs. Techniques such as DNA sequencing and PCR enable the identification of genetic mutations associated with various hereditary conditions. This allows for early diagnosis and the implementation of preventive measures or treatments to manage these conditions. Similar to dogs, cats also benefit from molecular diagnostics for the screening of
Bioscience Methods 2024, Vol.15, No.3, 91-101 http://bioscipublisher.com/index.php/bm 94 inherited diseases. Genetic testing can identify carriers of specific mutations, helping breeders make informed decisions and reducing the prevalence of genetic disorders in the feline population. 3.3 Oncology and cancer detection Molecular diagnostics are crucial for the early detection of tumors in pets. Liquid biopsy, which involves the analysis of circulating tumor DNA (ctDNA) or other tumor-derived molecules in body fluids, offers a non-invasive method for early cancer detection. This technique can identify tumors at an early stage, improving the chances of successful treatment. The identification of biomarkers through molecular diagnostics has transformed veterinary oncology. Biomarkers can indicate the presence of cancer, predict disease progression, and guide treatment decisions. The use of molecular tests to detect these biomarkers allows for personalized cancer therapy, improving outcomes for pets with cancer (Sokolenko and Imyanitov, 2018). 3.4 Monitoring and management of chronic diseases Molecular diagnostics are used to monitor and manage chronic diseases such as diabetes in pets. Genetic testing can identify predispositions to diabetes, while molecular assays can monitor glucose levels and other relevant biomarkers. This enables veterinarians to tailor treatment plans and manage the disease more effectively (Upadhyay et al., 2021). Chronic kidney disease (CKD) in pets can also be monitored using molecular diagnostics. Biomarkers identified through molecular techniques can indicate the early stages of kidney disease, allowing for timely intervention. Regular monitoring of these biomarkers helps in managing the progression of CKD and improving the quality of life for affected pets (Otto et al., 2016; Xue et al., 2021). By integrating molecular diagnostics into veterinary practice, the detection, treatment, and management of various diseases in pets have become more precise and effective, leading to better health outcomes and enhanced quality of life for our animal companions. 4 Technological Advancements in Molecular Diagnostics 4.1 Advances in PCR technology Polymerase Chain Reaction (PCR) technology has seen significant advancements, particularly with the integration of microfluidic systems. These systems have enabled the miniaturization of PCR processes onto chip devices, which offer benefits such as increased speed, reduced cost, enhanced portability, and automation. These improvements are crucial for point-of-care (POC) diagnostics, especially in resource-limited settings where traditional, centralized laboratory-based PCR methods are impractical (Park et al., 2011). Additionally, the development of loop-mediated isothermal amplification (LAMP) techniques linked to smartphone technology has further enhanced the efficiency and sensitivity of PCR-based diagnostics for various pathogens, including those affecting pets (Upadhyay et al., 2021). 4.2 Innovations in sequencing platforms Recent innovations in sequencing platforms have made significant strides in making molecular diagnostics more accessible and efficient. Mobile phone-based multimodal microscopes have been developed to perform targeted next-generation DNA sequencing and in situ mutation analysis. These portable devices allow for on-site diagnostics, which is particularly beneficial for telemedicine applications and remote veterinary care (Figure 2) (Kühnemund et al., 2017). Furthermore, the integration of plasmonic assays with sequencing technologies has facilitated the development of cost-effective, sensitive, and rapid diagnostic methods suitable for both developed and developing regions (Yu and Wei, 2018). 4.3 Integration of AI and machine learning The integration of artificial intelligence (AI) and machine learning into molecular diagnostics is revolutionizing the field by enhancing the accuracy and speed of disease detection. AI algorithms can analyze complex datasets generated by molecular diagnostic tools, providing rapid and precise diagnostic results. This integration is particularly useful in the development of portable diagnostic instruments, which require efficient data processing capabilities to function effectively in various settings, including veterinary clinics and field conditions (Lepej and Poljak, 2020). AI-driven platforms are also being used to improve the sensitivity and specificity of diagnostic assays, thereby reducing the likelihood of false positives and negatives.
Bioscience Methods 2024, Vol.15, No.3, 91-101 http://bioscipublisher.com/index.php/bm 95 Figure 2 Mobile phone microscopy-based targeted DNA sequencing (Adopted from Kühnemund et al., 2017) Image caption: (a) Amplified single-molecule detection through RCA and mobile phone microscopy: selected regions within images of 10 pM to 10 fM RCPs are depicted. Scale bar, 50 mm. (b) Quantification of RCPs generated from a log10 dilution series of synthetic circular templates. Error bars: 1 s.d. from the mean, n ¼ 3; linear regression is plotted as straight line. (c) KRAS wild type and (d) KRAS mutant (codon 12 mutation) synthetic DNA fragments were circularized through selector probes, amplified through RCA, and the first base in codon 12 sequenced by unchained SBL chemistry. Sequencing reactions were imaged at both of the fluorescent channels using the mobile phone microscope and these channels were digitally superimposed. Scale bar, 20 mm. (e) Synthetic KRAS fragments at a ratio of 1:1 000 mutant/wild type were sequenced and the reaction imaged through mobile phone microscopy. Cy3 stained RCPs (wild type-base G, green bar) and Cy5 stained RCPs (mutant-base A, red bar) were quantified and plotted in the inset graph. Red arrows in the mobile phone image point to RCPs that show a Cy5 sequencing signal. Scale bar, 200 mm. (f) Mobile phone microscopybased sequence analysis: (i) fluorescence imaging of sequencing reaction, and (ii) base calling through a custom-written automated image analysis algorithm. Scale bar, 50 mm. (g) Quantification of sequencing experiments of extracted genomic DNA from cell lines and colon tumour biopsies using our mobile phone microscope. Relative frequencies of mutant (red bars) and wild type (green bars) RCPs are plotted (Adopted from Kühnemund et al., 2017) 4.4 Portable and point-of-care diagnostic tools The development of portable and point-of-care (POC) diagnostic tools has been a game-changer in the field of molecular diagnostics. Advances in miniaturization, nanotechnology, and microfluidics have led to the creation of low-cost, user-friendly, and highly sensitive POC devices. These devices often integrate various biosensing platforms with smartphone technology, enabling accurate on-site diagnostics (Zarei et al., 2017). Additionally, paper-based nucleic acid testing (NAT) platforms have emerged as robust, cost-effective, and user-friendly tools for rapid diagnostics in resource-limited settings (Choi et al., 2015). The use of CRISPR-based diagnostics, such as the SHERLOCK platform, has further enhanced the portability and accuracy of POC tools, allowing for the detection of specific genetic signatures of pathogens directly from body fluids (Stower, 2018). In summary, the technological advancements in PCR technology, sequencing platforms, AI integration, and portable
Bioscience Methods 2024, Vol.15, No.3, 91-101 http://bioscipublisher.com/index.php/bm 96 diagnostic tools are collectively driving a new era in molecular diagnostics for pet disease detection. These innovations are making diagnostics more accessible, efficient, and accurate, ultimately improving the health and well-being of pets. 5 Challenges and Limitations 5.1 Technical challenges Molecular diagnostics in veterinary medicine have seen significant advancements, particularly in techniques such as PCR, gene sequencing, and mass spectrometry. However, these technologies come with technical challenges. Quality control is paramount to avoid issues such as inhibitors, cross-contamination, and inadequate templates, which can lead to erroneous microbial identifications. Additionally, the integration of clinical, pathologic, and laboratory findings is essential for accurate diagnosis, as molecular testing alone is insufficient. The development and implementation of these technologies also require thorough validation to ensure their reliability and accuracy in routine diagnostics (Cai et al., 2014). 5.2 Cost and accessibility The cost of molecular diagnostic tests remains a significant barrier to their widespread adoption in veterinary practice. The choice of technology and equipment, along with the need for specialized personnel training, contributes to the high costs associated with these tests. Furthermore, the reimbursement by third-party payers is often limited, making it challenging for veterinary clinics to justify the investment in these advanced diagnostic tools (Figure 3) (Fortina et al., 2002; Belák et al., 2013; Kostyusheva et al., 2020). The development of cost-effective alternatives and the potential for point-of-care testing through technologies like "lab-on-a-chip" could help mitigate these issues in the future. Figure 3 Schematics of CRISPR-Cas9-based CRISPR-diagnostic method CASLFA (Adopted from Kostyusheva et al., 2020) Image caption: (A) Structure of the lateral flow device. The lateral flow device consists of a sample pad where the isolate is applied, a conjugate pad with pre-assembled AuNP-DNA probes, a test line and a control line. At the test line, complexes of CRISPR-Cas with the target biotinylated DNA and AuNP-DNA probes, hybridized with the stem-loop region of sgRNA, interact with pre-coated streptavidin at the test pad to produce a visible signal. At the same time, AuNP-DNA probes move further and interact with streptavidin at the control line. AuNP-DNA probes contain three regions, namely (1) polyA-polyT (poly A used for labeling with Au and polyT as a linker); (2) purple area for hybridization with the embedded probe in the control line and (3) yellow area used for hybridization with the engineered stem-loop region in sgRNA. (B) Schematics of CASLFA procedure. Isolated DNA is amplified with biotinylated primers using RPA or PCR. Amplicons are mixed with CRISPR-Cas9 detection complex and DNA probes and, after short incubation, applied to the lateral flow device (Adopted from Kostyusheva et al., 2020)
Bioscience Methods 2024, Vol.15, No.3, 91-101 http://bioscipublisher.com/index.php/bm 97 5.3 Ethical considerations The use of molecular diagnostics in veterinary medicine raises several ethical considerations. One major concern is the potential for over-reliance on these technologies, which may lead to the neglect of traditional diagnostic methods and clinical judgment. Additionally, the handling and storage of genetic material pose privacy and biosecurity risks. Ensuring that these technologies are used responsibly and ethically is crucial to maintaining trust in veterinary diagnostics (Cai et al., 2014; Middleton et al., 2021). The balance between technological advancement and ethical practice must be carefully managed to avoid potential misuse or unintended consequences. 5.4 Regulatory hurdles Regulatory challenges are a significant obstacle to the implementation of molecular diagnostics in veterinary medicine. The approval process for new diagnostic tests can be lengthy and complex, often requiring extensive validation and demonstration of efficacy. Additionally, the regulatory landscape varies significantly between regions, complicating the global adoption of these technologies. Ensuring compliance with regulatory standards while fostering innovation is a delicate balance that must be achieved to facilitate the integration of molecular diagnostics into routine veterinary practice (Belák et al., 2009; Middleton et al., 2021). 6 Case Study 6.1 Description of the case A 7-year-old domestic short-haired cat presented with symptoms of lethargy, weight loss, and intermittent coughing. The cat had a history of outdoor activity and was not up-to-date on vaccinations. Initial physical examination revealed mild respiratory distress and palpable lymphadenopathy (Roest et al., 2017). 6.2 Diagnostic methods used To diagnose the underlying condition, a combination of molecular diagnostic techniques and imaging modalities were employed. Polymerase Chain Reaction (PCR) assays were conducted to detect potential viral pathogens. This included nested PCR for a range of DNA and RNA viruses, as well as real-time PCR for more accurate quantification and identification (Belák§ and Thorén, 2001). Positron Emission Tomography (PET) imaging was performed using 18F-FDG to assess metabolic activity in the lungs and lymph nodes. This method was chosen due to its high sensitivity in detecting early disease processes and differentiating between benign and malignant lesions (Rafiee et al., 2020; Phelps et al., 2000). Additionally, serological tests were conducted. Blood samples were analyzed for the presence of antibodies against common feline viruses to rule out other potential infections. 6.3 Outcomes and implications The PCR results identified the presence of a viral pathogen consistent with feline infectious peritonitis (FIP). PET/CT imaging revealed hypermetabolic activity in the lungs and mediastinal lymph nodes, indicative of an inflammatory or infectious process. The combination of these diagnostic methods allowed for a comprehensive assessment of the disease, confirming the diagnosis of FIP. The implications of this case are significant for veterinary practice. The use of advanced molecular diagnostics and imaging techniques facilitated early and accurate diagnosis, which is crucial for the management and treatment of infectious diseases in pets. This approach not only improves patient outcomes but also helps in implementing appropriate infection control measures to prevent the spread of the disease (Levin, 2008; Belák et al., 2009; Rafiee et al., 2020). 6.4 Lessons learned and future directions This case highlights several important lessons and future directions for the field of veterinary diagnostics. The integration of molecular diagnostics and imaging, such as the combination of PCR and PET/CT imaging, proved to be highly effective in diagnosing complex cases. Future research should focus on further integrating these technologies to enhance diagnostic accuracy and speed (Phelps, 2000). There is also a need for the continuous development of new diagnostic tools. The development of novel molecular probes and imaging assays to target specific pathogens and disease processes will improve the sensitivity and specificity of diagnostic methods (Levin, 2008). Standardization and harmonization are critical in this field. Efforts should be
Bioscience Methods 2024, Vol.15, No.3, 91-101 http://bioscipublisher.com/index.php/bm 98 made to standardize molecular diagnostic assays across different laboratories to ensure consistent and reliable results. International collaboration and adherence to guidelines will be essential in achieving this goal (Belák§ and Thorén, 2001). Training and education are equally important. Veterinary professionals should be trained in the use of advanced diagnostic technologies to fully leverage their potential in clinical practice. Ongoing education and training programs will be crucial in keeping up with technological advancements (Belák et al., 2009). By learning from this case and focusing on these future directions, the field of veterinary diagnostics can continue to evolve, leading to better health outcomes for pets and more effective disease management strategies. 7 Future Perspectives 7.1 Potential for personalized veterinary medicine The future of molecular diagnostics in veterinary medicine holds significant promise for personalized treatment plans tailored to individual animals. Advances in genomic and proteomic technologies are enabling the molecular subclassification of diseases, which can guide the selection of the most effective therapeutic agents. This approach mirrors the trends seen in human medicine, where single nucleotide polymorphisms and other genetic markers are used to predict disease predisposition and drug efficacy (Ross and Ginsburg, 2002; Jain et al., 2010). The integration of diagnostics with therapeutics is expected to enhance the precision of treatments, reducing side effects and improving outcomes for pets (Ross and Ginsburg, 2003). 7.2 Integration with telemedicine The integration of molecular diagnostics with telemedicine is another promising avenue. The ability to perform rapid, accurate diagnostic tests remotely can facilitate timely medical interventions, especially in areas lacking specialized veterinary services. This can be particularly beneficial for early disease detection and management, allowing veterinarians to provide expert care without the need for physical presence (Belák et al., 2009; Middleton et al., 2021). The shift from laboratory-based assays to patient-side diagnostics, enabled by technological advancements, will further support this integration. 7.3 The role of molecular diagnostics in preventive care Molecular diagnostics are poised to play a crucial role in preventive veterinary care. By identifying pathogens and disease markers before clinical symptoms appear, these technologies can help in the early intervention and management of diseases, thereby improving the overall health and longevity of pets (Belák et al., 2009). The ability to detect diseases at a molecular level allows for more effective monitoring and control of infectious diseases, which is essential for both individual animal health and broader public health concerns (Bonkobara, 2016). 7.4 Expected technological trends The field of molecular diagnostics is expected to continue evolving with several technological trends on the horizon. Innovations such as next-generation sequencing, advanced PCR techniques, and the use of biochips and microarrays are likely to become more prevalent in veterinary diagnostics. These technologies will enhance the accuracy, speed, and cost-effectiveness of diagnostic tests. Additionally, the development of non-invasive diagnostic tools and the use of nanobiotechnologies are expected to revolutionize the way diseases are detected and monitored in pets (Jain, 2010). The continuous improvement and adoption of these advanced diagnostic methods will undoubtedly shape the future landscape of veterinary medicine (Bonkobara, 2016). 8 Concluding Remarks The integration of molecular diagnostics into veterinary practice has revolutionized the detection and management of animal diseases. Technological advancements have expanded the array of diagnostic options, enabling faster and more accurate pathogen detection. Techniques such as PCR, nucleic acid probes, and hybridization studies are now commonplace, significantly enhancing the capabilities of veterinary laboratories. The development of real-time PCR and other advanced molecular tools has further improved the speed and sensitivity of diagnostics, allowing for precise identification and characterization of pathogens. These advancements not only facilitate timely responses to disease outbreaks but also support the implementation of effective biosafety and prophylactic measures.
Bioscience Methods 2024, Vol.15, No.3, 91-101 http://bioscipublisher.com/index.php/bm 99 The adoption of molecular diagnostics in veterinary practice has profound implications. It allows for the rapid and specific diagnosis of infectious, neoplastic, and congenital diseases, which is crucial for effective disease management and control. The ability to detect pathogens directly from clinical samples without the need for culture has streamlined the diagnostic process, making it more efficient and less reliant on traditional methods. Moreover, the integration of these advanced diagnostic tools into routine veterinary practice necessitates ongoing education and training for veterinarians to ensure they are well-versed in the use and interpretation of molecular diagnostic tests. This will enable practitioners to make informed decisions regarding disease management and treatment, ultimately improving animal health outcomes. Despite the significant advancements in molecular diagnostics, there is a continuous need for research to further refine and develop these technologies. Future research should focus on enhancing the sensitivity and specificity of diagnostic assays, as well as expanding their applicability to a broader range of pathogens and diseases. Additionally, there is a need to address the challenges associated with quality control and validation of molecular tests to ensure their reliability and accuracy in clinical settings. Collaborative efforts between veterinary researchers, diagnosticians, and practitioners will be essential in driving innovation and improving the overall effectiveness of molecular diagnostics in veterinary medicine. By investing in research and development, the veterinary field can continue to advance its diagnostic capabilities, ultimately leading to better disease control and improved animal health. Acknowledgments Authors are deeply grateful to the two anonymous peer reviewers for their insightful feedback on the manuscript. Conflict of Interest Disclosure Authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. Reference Abudayyeh O., and Gootenberg J., 2021, CRISPR diagnostics, Science, 372: 914-915. https://doi.org/10.1126/science.abi9335 Arora N., Chaudhary A., and Prasad A., 2023, Editorial: methods and applications in molecular diagnostics, Frontiers in Molecular Biosciences, 10: 5. https://doi.org/10.3389/fmolb.2023.1239005 Balamurugan V., Venkatesan G., Sen A., Annamalai L., Bhanuprakash V., and Singh R., 2010, Recombinant protein-based viral disease diagnostics in veterinary medicine, Expert Review of Molecular Diagnostics, 10: 731-753. https://doi.org/10.1586/erm.10.61 Belák S., Karlsson O., Blomström A., Berg M., and Granberg F., 2013, New viruses in veterinary medicine, detected by metagenomic approaches, Veterinary Microbiology, 165(1): 95-101. https://doi.org/10.1016/j.vetmic.2013.01.022 Belák S., Thorén P., Leblanc N., and Viljoen G., 2009, Advances in viral disease diagnostic and molecular epidemiological technologies, Expert Review of Molecular Diagnostics, 9: 367-381. https://doi.org/10.1586/erm.09.19 Belák§ S., and Thorén P., 2001, Molecular diagnosis of animal diseases: some experiences over the past decade, Expert Review of Molecular Diagnostics, 1: 434-443. https://doi.org/10.1586/14737159.1.4.434 Bonkobara M., 2016, Recent developments in veterinary diagnostics: current status and future potential, Veterinary Journal, 215(1-2): 16-19. https://doi.org/10.1016/j.tvjl.2016.08.010 Cai H., Caswell J., and Prescott J., 2014, Nonculture molecular techniques for diagnosis of bacterial disease in animals, Veterinary Pathology, 51: 341-350. https://doi.org/10.1177/0300985813511132 Choi J., Tang R., Wang S., Abas W., Pingguan-Murphy B., and Xu F., 2015, Paper-based sample-to-answer molecular diagnostic platform for point-of-care diagnostics, Biosensors & Bioelectronics, 74: 427-439. https://doi.org/10.1016/j.bios.2015.06.065 Daniels J., 2013, Molecular diagnostics for infectious disease in small animal medicine: an overview from the laboratory, The Veterinary Clinics of North America, Small Animal Practice, 43: 1373-1384. https://doi.org/10.1016/j.cvsm.2013.07.006 Fortina P., Surrey S., and Kricka L., 2002, Molecular diagnostics: hurdles for clinical implementation, Trends in Molecular Medicine, 8(6): 264-266. https://doi.org/10.1016/S1471-4914(02)02331-6
Bioscience Methods 2024, Vol.15, No.3, 91-101 http://bioscipublisher.com/index.php/bm 100 Granberg F., Karlsson O., Leijon M., Liu L., and Belák S., 2014, Molecular approaches to recognize relevant and emerging infectious diseases in animals, Veterinary Infection Biology: Molecular Diagnostics and High-Throughput Strategies, 1247: 109-124. https://doi.org/10.1007/978-1-4939-2004-4_7 Hatoum-Aslan A., 2018, CRISPR methods for nucleic acid detection herald the future of molecular diagnostics, Clinical Chemistry, 64(12): 1681-1683. https://doi.org/10.1373/clinchem.2018.295485 Jain K., 2010, Innovative diagnostic technologies and their significance for personalized medicine, Molecular Diagnosis & Therapy, 14: 141-147. https://doi.org/10.1007/BF03256366 Kaminski M., Abudayyeh O., Gootenberg J., Zhang F., and Collins J., 2021, CRISPR-based diagnostics, Nature Biomedical Engineering, 5: 643-656. https://doi.org/10.1038/s41551-021-00760-7 Kostyusheva A., Brezgin S., Babin Y., Vasil’eva I., Kostyushev D., and Chulanov V., 2020, CRISPR-cas systems for diagnosing infectious diseases, Methods (San Diego, Calif.), 203: 431-446. https://doi.org/10.1016/j.ymeth.2021.04.007 Kühnemund M., Wei Q., Darai E., Wang Y., Hernández-Neuta I., Yang Z., Tseng D., Ahlford A., Mathot L., Sjöblom T., Ozcan A., and Nilsson M., 2017, Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy, Nature Communications, 8: 19-23. https://doi.org/10.1038/ncomms13913 Kurkela S., and Brown D., 2009, Molecular diagnostic techniques, Medicine (Abingdon, England : UK Ed.), 37: 535-540. https://doi.org/10.1016/j.mpmed.2009.07.012 Lappin M., 2009, Infectious disease diagnostic assays, Topics in Companion Animal Medicine, 24: 199-208. https://doi.org/10.1053/j.tcam.2009.07.004 Lepej S., and Poljak M., 2020, Portable molecular diagnostic instruments in microbiology: current status, Clinical Microbiology and Infection : The Official Publication of The European Society of Clinical Microbiology and Infectious Diseases, 9: 17. https://doi.org/10.1016/j.cmi.2019.09.017 Levin C., 2008, New imaging technologies to enhance the molecular sensitivity of positron emission tomography, Proceedings of The IEEE, 96: 439-467. https://doi.org/10.1109/JPROC.2007.913504 Middleton J., Getchell R., Flesner B., Hess W., Johnson P., Scarfe A., and Starling D., 2021, Considerations related to the use of molecular diagnostic tests in veterinary clinical and regulatory practice, Journal of the American Veterinary Medical Association, 259(6): 590-595. https://doi.org/10.2460/javma.259.6.590 Middleton J., Getchell R., Flesner B., Hess W., Johnson P., Scarfe A., and Starling D., 2021, Considerations related to the use of molecular diagnostic tests in veterinary clinical and regulatory practice, Journal of the American Veterinary Medical Association, 259(6): 590-595. https://doi.org/10.2460/javma.259.6.590 Otto C., Angle T., Passler T., Waggoner P., Fischer T., Rogers B., Galik P., and Maxwell H., 2016, Real-time detection of a virus using detection dogs, Frontiers in Veterinary Science, 2: 79. https://doi.org/10.3389/fvets.2015.00079 Park S., Zhang Y., Lin S., Wang T., and Yang S., 2011, Advances in microfluidic PCR for point-of-care infectious disease diagnostics, Biotechnology Advances, 29(6): 830-839. https://doi.org/10.1016/j.biotechadv.2011.06.017 Phelps M., 2000, PET: the merging of biology and imaging into molecular imaging, journal of nuclear medicine : official publication, Society of Nuclear Medicine, 41(4): 661-681. Rafiee F., Keshavarz P., Katal S., Assadi M., Nejati S., Sadabad F., and Gholamrezanezhad A., 2020, Coronavirus disease 2019 (covid-19) in molecular imaging: a systematic review of incidental detection of sars-cov-2 pneumonia on pet studies, Seminars in Nuclear Medicine, 51: 178-191. https://doi.org/10.1053/j.semnuclmed.2020.10.002 Roest H., Engelsma M., Weesendorp E., Bossers A., and Elbers A., 2017, Veterinary molecular diagnostics, Seminars in Nuclear Medicine, 11: 219-234. https://doi.org/10.1007/978-981-10-4511-0_11 Ross J., and Ginsburg G., 2002, Integration of molecular diagnostics with therapeutics: implications for drug discovery and patient care, Expert Review of Molecular Diagnostics, 2: 531-541. https://doi.org/10.1586/14737159.2.6.531 Sala A., and Perani D., 2019, Brain molecular connectivity in neurodegenerative diseases: recent advances and new perspectives using positron emission tomography, Frontiers in Neuroscience, 13: 78-90. https://doi.org/10.3389/fnins.2019.00617 Shahi S., Vahed S., Fathi N., and Sharifi S., 2018, Polymerase chain reaction (PCR)-based methods: Promising molecular tools in dentistry, International Journal of Biological Macromolecules, 117: 983-992. https://doi.org/10.1016/j.ijbiomac.2018.05.085 Sokolenko A., and Imyanitov E., 2018, Molecular diagnostics in clinical oncology, Frontiers in Molecular Biosciences, 5: 8. https://doi.org/10.3389/fmolb.2018.00076 Stower H., 2018, CRISPR-based diagnostics, Nature Medicine, 24: 702-702. https://doi.org/10.1038/s41591-018-0073-z Upadhyay A., Waleed R., Wang J., Zhao J., Guan Q., Liao C., and Han Q., 2021, An innovative and user-friendly smartphone-assisted molecular diagnostic approach for rapid detection of canine vector-borne diseases, Parasitology Research, 2: 5.
Bioscience Methods 2024, Vol.15, No.3, 91-101 http://bioscipublisher.com/index.php/bm 101 Weng Z., You Z., Yang J., Mohammad N., Lin M., Wei Q., Gao X., and Zhang Y., 2023, CRISPR-cas biochemistry and CRISPR-based molecular diagnostics, Angewandte Chemie, 5: 67. https://doi.org/10.1002/anie.202214987 Wright W., Simner P., Carroll K., and Auwaerter P., 2021, Progress report: next-generation sequencing (NGS), multiplex polymerase chain reaction (PCR), and broad-range molecular assays as diagnostic tools for fever of unknown origin (FUO) investigations in adults, Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America, 34: 155. https://doi.org/10.1093/cid/ciab155 Xue Y., Kong Q., Ding H., Xie C., Zheng B., Zhuo X., Ding J., Tong Q., Lou D., Lu S., and Lv H., 2021, A novel loop-mediated isothermal amplification-lateral-flow-dipstick (lamp-lfd) device for rapid detection of toxoplasma gondii in the blood of stray cats and dogs, Parasite, 28: 56-58. https://doi.org/10.1051/parasite/2021039 Yin K., Ding X., Li Z., Zhao H., Cooper K., and Liu C., 2020, Dynamic aqueous multiphase reaction system for one-pot CRISPR-Cas12a based ultrasensitive and quantitative molecular diagnosis, Analytical Chemistry, 2: 45-67. https://doi.org/10.1021/acs.analchem.0c01459 Yu T., and Wei Q., 2018, Plasmonic molecular assays: recent advances and applications for mobile health, Nano Research, 11: 5439-5473. https://doi.org/10.1007/s12274-018-2094-9 Zarei M., 2017, Advances in point-of-care technologies for molecular diagnostics, Biosensors & bioelectronics, 98: 494-506. https://doi.org/10.1016/j.bios.2017.07.024
Bioscience Methods 2024, Vol.15, No.3, 102-113 http://bioscipublisher.com/index.php/bm 102 Research Report Open Access Transcriptomic Approaches to Studying Rice Pathogen Interactions Yuming Huang School of Life Sciences, Xiamen University, Xiamen, 361102, Fujian, China Corresponding email: hym@xmu.edu.cn Bioscience Methods, 2024, Vol.15, No.3 doi: 10.5376/bm.2024.15.0012 Received: 29 Mar., 2024 Accepted: 22 May, 2024 Published: 02 Jun., 2024 Copyright © 2024 Huang, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Huang Y.M., 2024, Molecular diagnostics: a new era in pet disease detection, Bioscience Methods, 15(3): 102-113 (doi: 10.5376/bm.2024.15.0012) Abstract Understanding the intricate interactions between rice (Oryza sativa) and its pathogens is crucial for developing effective disease management strategies. Transcriptomic approaches have significantly advanced our knowledge in this area by enabling comprehensive profiling of gene expression during infection. This study leverages high-quality RNA sequencing and other transcriptomic techniques to explore the dynamic interactions between rice and various pathogens, including the rice blast fungus (Magnaporthe oryzae) and the Rice black-streaked dwarf virus (RBSDV). Key findings include the identification of differentially expressed mRNAs and long non-coding RNAs (lncRNAs) that play essential roles in rice's defense mechanisms, as well as novel microRNAs (miRNAs) that regulate pathogen resistance genes. Additionally, tissue-specific expression patterns of pathogenicity genes and miRNAs were observed, providing deeper insights into the dual-epidemics of blast disease. These transcriptomic analyses offer a valuable resource for understanding the molecular mechanisms underlying rice-pathogen interactions and pave the way for developing improved disease-resistant rice varieties. Keywords Rice-pathogen interactions; Transcriptomics; Magnaporthe oryzae; Long non-coding RNAs (lncRNAs); MicroRNAs (miRNAs) 1 Introduction Rice (Oryza sativa L.) is a staple food crop that feeds more than half of the world's population. However, the stability and growth of rice yield are facing threats from a variety of biotic and abiotic factors (Yang, 2024). Among them, various pathogens, including bacteria, fungi, and viruses, which lead to significant yield losses. Bacterial blight (BB) and bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) respectively, are particularly detrimental (Jiang et al., 2020). Additionally, fungal diseases such as rice blast, caused by Magnaporthe oryzae, and other fungal pathogens like Pyricularia oryzae, Ustilaginoidea virens, and Rhizoctonia solani, pose severe threats to rice crops globally (Liu et al., 2014; He et al., 2022). Understanding the interactions between rice and these pathogens is crucial for developing effective disease management strategies. The study of transcriptomic responses in rice during pathogen interactions has provided significant insights into the molecular mechanisms underlying plant defense and pathogen attack. Transcriptomics, which involves the comprehensive analysis of RNA transcripts, allows researchers to identify infection-responsive genes and understand their roles in disease resistance and susceptibility (Sarki et al., 2020). For instance, transcriptomic analyses have revealed the upregulation of specific genes in rice that are involved in defense responses, such as pathogenesis-related proteins and phytoalexin biosynthetic genes, during interactions with pathogens (Kawahara et al., 2012). Moreover, the identification of pathogen-associated molecular patterns (PAMPs) and effector-triggered immunity (ETI) has advanced our understanding of the complex signaling networks that govern plant immunity (Liu et al., 2014; Liu and Wang, 2016). These insights are essential for developing new strategies to enhance disease resistance in rice through genetic and genomic approaches. This study utilizes transcriptomic approaches to investigate the interactions between rice and its pathogens. By analyzing the gene expression profiles of both rice and its pathogens during infection, this study identifies key genes and pathways involved in the defense response and pathogen virulence; focuses on the characterize of the transcriptomic changes in rice during interactions with bacterial and fungal pathogens; identifies and analyzes the
Bioscience Methods 2024, Vol.15, No.3, 102-113 http://bioscipublisher.com/index.php/bm 103 expression of infection-responsive genes in both rice and pathogens, and explores the potential roles of identified genes in disease resistance and susceptibility. This study aims to provide insights into the molecular mechanisms underlying rice-pathogen interactions that can inform the development of disease-resistant rice varieties. 2 Overview of Rice Pathogens 2.1 Major rice pathogens and their economic impact Rice (Oryza sativa L.) is a staple food crop for more than half of the world's population, making it a critical component of global food security. However, rice production is severely threatened by various pathogens, including bacteria, fungi, viruses, and nematodes, which can lead to significant yield losses. Among the bacterial pathogens, Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) are responsible for bacterial blight (BB) and bacterial leaf streak (BLS), respectively, both of which are major diseases affecting rice production worldwide (Khojasteh et al., 2017; Jiang et al., 2020). Fungal pathogens such as Magnaporthe oryzae, which causes rice blast disease, also pose a significant threat, leading to substantial yield reductions (Liu and Wang, 2016). Additionally, the root-knot nematode (RKN), Meloidogyne graminicola, is known to cause extensive yield decline in rice crops (Kumari et al., 2016). The economic impact of these pathogens is profound, as they not only reduce grain yield but also affect the quality of the produce, thereby threatening the livelihood of millions of farmers and the food supply for billions of people (Kazan and Gardiner, 2018; Wei, et al., 2023). 2.2 Host-pathogen interaction mechanisms in rice Understanding the mechanisms of host-pathogen interactions in rice is essential for developing effective disease management strategies. Rice has evolved a multi-layered immune system to combat pathogen invasion. This includes pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is initiated upon recognition of conserved microbial molecules, while ETI is activated by specific pathogen effectors recognized by resistance (R) genes in the host (Costa, 2012; Liu and Wang, 2016). For instance, the interaction between rice and Xoo involves a series of R genes and their corresponding avirulence (Avr) genes, which play a crucial role in the plant's defense response (Khojasteh et al., 2017; Jiang et al., 2020). Similarly, proteomic studies have identified several proteins involved in rice's defense against Magnaporthe oryzae, including receptor-like kinases (RLKs) and mitogen-activated protein kinases (MAPKs), which are pivotal in signal transduction and defense response. Hormonal pathways, such as those regulated by salicylate, jasmonate, and ethylene, also play significant roles in modulating the plant's defense mechanisms against various pathogens (Kumari et al., 2016). 2.3 Traditional methods for studying rice pathogen interactions Traditional methods for studying rice-pathogen interactions have primarily focused on genetic and genomic approaches. These include the identification and characterization of R genes and their corresponding Avr genes, as well as the use of quantitative trait loci (QTL) mapping to understand the genetic basis of disease resistance (Jiang et al., 2020; Roychoudhury, 2020). Microarray and gene expression studies have been employed to identify differentially expressed genes (DEGs) in response to pathogen infection, providing insights into the molecular mechanisms underlying host-pathogen interactions (Khojasteh et al., 2017). Proteomic analyses have also been instrumental in identifying key proteins involved in the plant's defense response, thereby enhancing our understanding of the complex interactions between rice and its pathogens (Meng et al., 2019; Wei, et al., 2023). These traditional methods have laid the foundation for more advanced transcriptomic and proteomic studies, which continue to unravel the intricate details of rice-pathogen interactions and aid in the development of disease-resistant rice varieties. 3 Transcriptomic Approaches 3.1 Introduction to transcriptomics and its relevance in plant-pathogen studies Transcriptomics, the study of the complete set of RNA transcripts produced by the genome under specific circumstances, has become a pivotal tool in understanding plant-pathogen interactions. This field allows researchers to capture a snapshot of gene expression at a given time, providing insights into the molecular mechanisms underlying these interactions. By analyzing the transcriptome, scientists can identify which genes
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