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Molecular identification of haemoparasites in animals using blood lysate PCR: a quick and inexpensive alternative to purified whole genomic DNA.
Abstract: Haemoparasitic diseases constitute a significant constraint to economic livestock farming. Diagnostic techniques that are inexpensive, rapid, reliable, and precise are crucial for the management of diseases. In this context, PCR assays are very valuable yet expensive since the samples must be processed before being included in the PCR reaction. Accordingly, the goal of the current study was to lower the PCR costs without jeopardizing the assay's sensitivity and specificity. For that purpose, the alkaline solution was optimized for low cost and quick DNA extraction (blood lysate), and PCR reagents were modified for optimum reaction. In comparison to purified whole blood genomic DNA, the currently developed and optimized blood lysate method was found to be 95.5% less expensive, as well as being equally sensitive and specific for the molecular detection (PCR) of haemoparasites like , and rickettsiales in cattle, buffaloes, horses, and dogs. The assay was also demonstrated to be quick, less likely to cross-contaminate, and appropriate for use in laboratories with limited resources. Therefore, the currently developed and optimized blood lysate method could serve as a viable alternative to purified whole blood genomic DNA for molecular detection (PCR) of haemoparasites in animals particularly in resource-limited settings.
Publication Date: 2024-08-13 PubMed ID: 39136443PubMed Central: PMC12674356DOI: 10.1080/10495398.2024.2390935Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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Overview
- This study developed and optimized a rapid, inexpensive PCR method using blood lysate instead of purified whole genomic DNA to detect haemoparasitic infections in animals.
- The optimized method maintains high sensitivity and specificity while drastically reducing the cost and processing time, making it suitable for resource-limited laboratories.
Background
- Haemoparasitic diseases are a major economic burden on livestock farming globally due to their impact on animal health and productivity.
- Accurate, rapid, and affordable diagnostic methods are essential for effective management and control of these infections.
- PCR (Polymerase Chain Reaction) techniques are highly valuable for detecting haemoparasites at the molecular level but traditionally require purified genomic DNA from blood samples.
- DNA purification steps tend to be time-consuming, labor-intensive, and costly, limiting PCR use in many field or resource-poor settings.
Goals of the Study
- To develop a quick and cost-effective method for DNA preparation directly from blood samples without full genomic DNA purification.
- To optimize an alkaline lysis solution that effectively releases DNA into a blood lysate suitable for PCR.
- To adjust PCR reagents and conditions to maintain sensitivity and specificity equivalent to those achieved with purified DNA templates.
- To validate this new approach across a range of haemoparasite species and animal hosts important in veterinary practice.
Methods
- Blood samples were collected from animals such as cattle, buffaloes, horses, and dogs potentially infected with haemoparasites.
- An alkaline solution was optimized for lysing blood cells quickly and cost-effectively to release parasite DNA without the need for DNA purification kits.
- PCR reagent concentrations and cycling parameters were modified to maximize amplification success using blood lysate as the template.
- The assay’s performance was compared to standard PCR using purified whole genomic DNA as the template.
- Parasites targeted in the assay included common haemoparasites and rickettsial pathogens relevant to livestock health.
Findings
- The blood lysate PCR method reduced the cost by approximately 95.5% compared to conventional PCR requiring purified DNA.
- The sensitivity (ability to detect true positives) and specificity (ability to avoid false positives) of PCR with blood lysate were comparable to those using purified genomic DNA.
- The new method considerably decreased assay processing time, facilitating quicker diagnosis.
- Due to the streamlined process, the assay lowered the risk of cross-contamination, an important consideration in diagnostic labs.
- The method was versatile across multiple haemoparasite species and different animal hosts, indicating broad applicability.
- Its simplicity and low resource requirements make it ideal for laboratories with limited infrastructure or funding.
Implications
- This optimized blood lysate PCR method offers a practical alternative to the standard purified DNA PCR assays for haemoparasite diagnosis.
- It enables faster, cheaper, and reliable detection of haemoparasites, which is especially vital in low-resource or field conditions.
- By lowering barriers to molecular diagnostics, the method can improve surveillance, disease management, and control programs in veterinary settings.
- Additional validation and adaptation could expand its use to other infectious diseases in veterinary and potentially human medicine.
Conclusion
- The study successfully demonstrated that PCR using an optimized blood lysate preparation is a sensitive, specific, quick, and cost-efficient alternative to purified DNA PCR for detecting haemoparasites.
- This method is well-suited for resource-limited laboratories aiming to enhance diagnostic capacity for economically significant haemoparasitic diseases in animals.
Cite This Article
APA
Kumar B, Brahmbhatt NN, Thakre B, Maharana BR, Parmar VL, Kumar M.
(2024).
Molecular identification of haemoparasites in animals using blood lysate PCR: a quick and inexpensive alternative to purified whole genomic DNA.
Anim Biotechnol, 35(1), 2390935.
https://doi.org/10.1080/10495398.2024.2390935 Publication
Researcher Affiliations
- Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Junagadh, India.
- Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Junagadh, India.
- Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Junagadh, India.
- Referral Veterinary Diagnostic and Extension Centre, LUVAS, Karnal, India.
- Department of Veterinary Medicine, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Junagadh, India.
- Central Animal House, Indira Gandhi Institute of Medical Sciences, Patna, India.
MeSH Terms
- Animals
- Polymerase Chain Reaction / methods
- Polymerase Chain Reaction / veterinary
- Buffaloes
- Cattle
- Horses
- Dogs
- Babesia / isolation & purification
- Babesia / genetics
- Sensitivity and Specificity
- Trypanosoma / isolation & purification
- Trypanosoma / genetics
- DNA, Protozoan / genetics
- Theileria / isolation & purification
- Theileria / genetics
- DNA / blood
- DNA / isolation & purification
- Cattle Diseases / diagnosis
- Cattle Diseases / parasitology
- Cattle Diseases / blood
- Dog Diseases / blood
Conflict of Interest Statement
No potential conflict of interest was reported by the author(s).
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