Loop-Mediated Isothermal Amplification: Pathogen Detection in Veterinary, Food, and Water Samples

Isothermal nucleic acid amplification is a versatile and powerful tool in both human and veterinary diagnostics, offering rapid, sensitive, and specific detection of nucleic acids. Some of the key applications include:

• Infectious disease detection: Rapid identification of bacterial, viral, and parasitic infections
• Genetic testing: Detection of genetic mutations and SNPs
• Cancer diagnostics: Identifying specific biomarkers associated with different types of cancer

LAMP using Bst DNA polymerase is robust, rapid, simple, cost-effective, and an excellent option for several diagnostic applications. Additionally, for organizations moving into the process development and GMP manufacturing phases, LAMP can be a cell-free test of choice, which cuts down costly quality testing and additional analytical tests that survey for animal or bacterial contaminants in the amplified DNA template. DNA fragments amplified by LAMP can be visually assessed by fluorescence visible to the naked eye or by gel electrophoresis methods (Figure 1). The versatility, relative simplicity, and robustness make LAMP an excellent candidate for non-clinical applications as well. This article highlights the successes using LAMP technique employing Bst polymerase in non-clinical applications such as pathogen detection in insect vectors, agriculture, livestock, environment, and food testing. 

In addition to viroids, fungal and fungal-like pathogens are a threat to food crops. RT-LAMP with Superscript IV RT was used to detect the fungi Botrytis cinerea and Fusarium graminearum and oomycetes Phytophthora cactorum and Phytophthora plurivora. Similar methods were used to detect the fungi Botrytis cinerea and Fusarium graminearum along with the oomycetes Phytophthora cactorum and Phytophthora plurivora.

Invasive insect pests also pose a threat to our food production systems. For example, an invasive food pest Spodoptera frugiperda was introduced from the United States to Africa, an area already rife with food security challenges and public health threats. LAMP can be used to detect S. frugiperda in the field or when specimen collection conditions make species identification by eye challenging or impossible [1]. The minimal equipment needs and user-friendliness of LAMP in the field allows surveyors to test samples right away in an area where potentially invasive strains of S. frugiperda are present. In addition to agricultural pests, public health threats like mosquitoes can be identified at ports of entry using Bst DNA polymerase and LAMP in a similar way to S. frugiperda, allowing almost same-day identification and control because of the rapidity, sensitivity, and specificity of the LAMP assay.

In the veterinary space, large animals suffer from viral and epizootic diseases that could result in the culling of livestock and straining our food supply chain. LAMP with Bst DNA polymerase was used to detect 18 notifiable viral diseases of large animals by the World Organization for Animal Health [2]. These diseases included Bluetongue virus, Rift Valley fever, Swine fever, and Avian influenza. Early detection of pathogens aids in timely quarantine measures, which help prevent the establishment and transmission of disease. LAMP with Bst DNA Polymerase provides accessibility to rapid, sensitive, and specific testing for the livestock industry. For our furry friends, screening for cystic echinococcus in dogs using LAMP provided greater sensitivity and specificity compared to PCR and ELISA [3].

Environmental sampling of pathogens is important for reducing the burden of disease on society. Clean water supply is essential for human health and safety; but water is always at risk for contamination with fecal coliforms and other waterborne pathogens. Presence or absence of fecal coliforms—such as Enterococcus species—are used as indicators of water quality. 

In the past, the most trusted water quality testing method was sample filtration followed by agar plating of the water specimen [4]. Despite the success of this method, faster testing methods with equivalent robustness and sensitivity was needed to meet the increasing demand of water quality testing. qPCR replaced the culture method for the detection of environmental pathogens. However, due to constraints of specialty equipment and laboratory space requirements, qPCR is difficult to use in the field. 

LAMP technology offers a simple, accurate, and robust method for testing water quality in the field without the burden of cost, equipment, and expertise. Recent advantages in centrifugal microfluidic chip technology combined with LAMP is a promising solution to rapid field detection of waterborne pathogens [5].

According to Kundapur and Nema, LAMP can be used to identify GMOs by targeting specific DNA sequences that are unique to the genetic modification [6]. The two non-clinical applications that are of particular interest to the food industry are: identification of genetically modified organisms (GMOs) and meat product testing. LAMP enables the rapid and sensitive detection of genetically modified crops using LAMP by screening for its exogenous genes [6]. LAMP assays can be designed to detect and differentiate between different types of GMOs [7], offering a reliable means of identifying the presence of genetically modified agricultural products for regulatory compliance and ensuring transparency in the food chain. LAMP assays have been used for rapid detection of foreign genes in transgenic crops [8].

LAMP is also utilized in meat quality testing. Often, a package of animal products may be mislabeled as one species, while containing a completely different species or a mixture of various meats, all unbeknownst to the consumer [9]. Assay sensitivity offered by the Bst DNA polymerase in the LAMP process is key for detecting food adulteration in large quantities of ground or cooked meat product. An example of how this is utilized in the meat market is through the identification of chicken meat contaminating red meat products [10]. The authors developed a LAMP assay targeting the chicken mitochondrial 16s rRNA gene, and their assay achieved high sensitivity and specificity for the detection of chicken in red meat in 30 minutes [10].