Comparison of DNA extraction methods for molecular identification of pathogenic Leptospira in the urine samples

Abstract

Latar belakang: Leptospirosis merupakan zoonosis penting di dunia, yang masih sering terjadi salah diagnosis. Deteksi laboratorium Leptospira menjadi tantangan karena bakterimea cukup singkat untuk dideteksi molekuler, namun antibodi juga muncul sangat lambat. Urine dapat menjadi sampel alternatif untuk deteksi PCR pada leptospirosis. Pengerjaan PCR membutuhkan DNA berkualitas dan andal, dan diperoleh dari metode ekstraksi DNA yang baik. Penelitian bertujuan untuk mengetahui metode ekstraksi DNA Leptospira terbaik untuk sampel urin, serta mengevaluasi pengaruh waktu penyimpanan dan suhu terhadap kestabilan DNA.
 Metode: Penelitian ini menggunakan tiga metode isolasi DNA yang berbeda; berbasis silika dengan spin kolom, kromatografi spin column menggunakan resin sebagai matriks pemisah, dan metode larutan dengan guanidine isothiocyanate. Hasil ekstraksi diperiksa konsentrasi dan kemurniannya. Gen SecY pada Leptospira dideteksi dengan PCR real-time. Pengaruh suhu dan lama penyimpanan DNA juga dilihat.
 Hasil: Hasil isolasi DNA menggunakan resin menunjukkan konsentrasi tertinggi (7,94 + 2,11 μg / mL) dan jumlah salinan amplifikasi DNA Leptospira tertinggi (50167,92 + 1,19). Suhu penyimpanan pada suhu 4°C, -20°C, dan -80°C dan umur simpan 91 hari tidak berpengaruh terhadap kualitas dan kuantitas DNA Leptospira hasil isolasi spike urin.
 Kesimpulan: Isolasi DNA menggunakan spin column chromatography dengan resin sebagai matriks separasi memiliki kualitas dan kuantitas terbaik berdasarkan kemurnian dan konsentrasi DNA serta jumlah gen SecY yang teramplifikasi.
 Kata kunci: Leptospira, Leptospirosis, ekstraksi DNA, sampel urin, penyimpanan sampel.
 
 Abstract
 Background: Leptospirosis is a worldwide zoonotic disease, which is still often misdiagnosed. Laboratory detection of Leptospira is challenging since the bacteraemia is quite short for molecular detection, however, the rise of the antibody is late to post the infection. Urine can be a potential alternative sample for PCR detection in leptospirosis. The PCR method requires a reliable DNA template, which is obtained from good DNA extracting methods. The study aimed to determine the best method of extraction Leptospira DNA from the urine sample, as well as evaluating the effect of time storage and temperature for its DNA stability.
 Methods: This study was utilizing three different DNA isolation methods; silica based with spin column, spin column chromatography using resin as separation matrix, and solution method with guanidine isothiocyanate. The yields were examined for its concentration and purity. Leptospira’s SecY gene was detected with realtime PCR. The influences of storage temperature and the life time of the DNA were also studied.
 Results: The yield of DNA isolation using resin showed the highest concentration (7.94+2.11 μg/mL) and highest Leptospira DNA amplification copy number (50167.92+1.19). Storage temperature at 4°C, -20°C, and -80°C and life time of 91 days did not have any effect on the quality and quatnity of Leptospira DNA isolated from spiked urine.
 Conclusions: DNA isolation using spin column chromatography with resin as separation matrix has the best quality and quantity based on the purity and concentration of DNA and the higher number of amplified SecY gene.
 Keywords: Leptospira, Leptospirosis, DNA extraction, urine sample, sample storage

Molecular Diagnosis as an Alternative for Public Health Surveillance of Leptospirosis in Colombia

“…The sensitivity of the amplification method is highly dependent on the extraction methods employed [73,75]. The reduction in the cost and availability of rapid extraction methods is a determining factor.…”

“…Real-time PCR detection with probes or intercalants is more sensitive, timely, and less susceptible to contamination than the conventional variant (Table 1), detecting between 102 to 103 leptospires per mL and achieving an analytical sensitivity of 1 to 50 copies in acute phase serum or plasma samples [71]. Taking into account the different extraction methods used, it has been proposed that the presence of inhibitors in urine samples may affect the reproducibility, resulting in a lower detection limit [24,75]. Positive results can be obtained from samples containing as little as 10 copies [73].…”