Development of Electrochemical DNA Biosensor for Equine Hindgut Acidosis Detection.

Joshua Davies,Carol Thomas,Mohammad Rizwan,Christopher Gwenin

doi:10.3390/s21072319

Joshua Davies, Carol Thomas + Show 2 more

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https://doi.org/10.3390/s21072319

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Abstract

The pH drop in the hindgut of the horse is caused by lactic acid-producing bacteria which are abundant when a horse’s feeding regime is excessively carbohydrate rich. This drop in pH below six causes hindgut acidosis and may lead to laminitis. Lactic acid-producing bacteria Streptococcus equinus and Mitsuokella jalaludinii have been found to produce high amounts of L-lactate and D-lactate, respectively. Early detection of increased levels of these bacteria could allow the horse owner to tailor the horse’s diet to avoid hindgut acidosis and subsequent laminitis. Therefore, 16s ribosomal ribonucleic acid (rRNA) sequences were identified and modified to obtain target single stranded deoxyribonucleic acid (DNA) from these bacteria. Complementary single stranded DNAs were designed from the modified target sequences to form capture probes. Binding between capture probe and target single stranded deoxyribonucleic acid (ssDNA) in solution has been studied by gel electrophoresis. Among pairs of different capture probes and target single stranded DNA, hybridization of Streptococcus equinus capture probe 1 (SECP1) and Streptococcus equinus target 1 (SET1) was portrayed as gel electrophoresis. Adsorptive stripping voltammetry was utilized to study the binding of thiol modified SECP1 over gold on glass substrates and these studies showed a consistent binding signal of thiol modified SECP1 and their hybridization with SET1 over the gold working electrode. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to examine the binding of thiol modified SECP1 on the gold working electrode and hybridization of thiol modified SECP1 with the target single stranded DNA. Both demonstrated the gold working electrode surface was modified with a capture probe layer and hybridization of the thiol bound ssDNA probe with target DNA was indicated. Therefore, the proposed electrochemical biosensor has the potential to be used for the detection of the non-synthetic bacterial DNA target responsible for equine hindgut acidosis.

Highlights

An excess of carbohydrates in the diet can lead to the onset of laminitis in horses, a condition where the distal phalanx and the inner hoof wall fail to attach, resulting in damage to the hoof and the surrounding area [1]
This study demonstrated that only one out of the four initial target and capture probe combinations, Streptococcus equinus capture probe 1 (SECP1)/Streptococcus equinus target 1 (SET1), were able to hybridize successfully
This is shown by polyacrylamide gel electrophoresis gels where each of the combinations were tested for the hybridization

Summary

Introduction

An excess of carbohydrates in the diet can lead to the onset of laminitis in horses, a condition where the distal phalanx and the inner hoof wall fail to attach, resulting in damage to the hoof and the surrounding area [1]. Failed attachment causes the distal phalanx to be forced down into the sole of the hoof under the horse’s weight [2] This leads to lameness, and often euthanasia of the horse as a result [1,2]. 7–14% of horses worldwide and is usually diagnosed from the observation of certain behavioral patterns such as an unwillingness to walk, uneven distribution of weight, and lameness [3]. Racehorses, in particular, suffer from metabolic issues because of their dietary setup which consists of short bursts of concentrate feeding in between lengthier periods of grazing [5] This carbohydrate-intensive feeding regime can cause the bacterial population in the hindgut to change as quickly as five hours post concentrate feed leading to equine hindgut acidosis and other similar metabolic problems [5]. As the pH drops below six, the horse can be diagnosed with clinical hindgut acidosis [5]

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