Freeze-Thaw Pretreatment Can Improve Efficiency of Bacterial DNA Extraction From Meconium.

Yuntian Xin,Xueqin Zhang,Jingxian Xie,Yunshan Xiao,Heqing Shen,Quanfeng Wu,Yi Lin,Bingru Nan,Chen Tang

doi:10.3389/fmicb.2021.753688

Abstract

Although the presence of live microbes in utero remains under debate, newborn gastrointestinal bacteria are undoubtedly important to infant health. Measuring bacteria in meconium is an ideal strategy to understand this issue; however, the low efficiency of bacterial DNA extraction from meconium has limited its utilization. This study aims to improve the efficiency of bacterial DNA extraction from meconium, which generally has low levels of microflora but high levels of PCR inhibitors in the viscous matrix. The research was approved by the ethical committee of the Xiamen Maternity and Child Health Care Hospital, Xiamen, China. All the mothers delivered naturally, and their newborns were healthy. Meconium samples passed by the newborns within 24 h were collected. Each sample was scraped off of a sterile diaper, transferred to a 5-ml sterile tube, and stored at −80°C. For the assay, a freeze-thawing sample preparation protocol was designed, in which a meconium-InhibitEX buffer mixture was intentionally frozen 1–3 times at −20°C, −80°C, and (or) in liquid nitrogen. Then, DNA was extracted using a commercial kit and sequenced by 16S rDNA to verify the enhanced bacterial DNA extraction efficiency. Ultimately, we observed the following: (1) About 30 mg lyophilized meconium was the optimal amount for DNA extraction. (2) Freezing treatment for 6 h improved DNA extraction at −20°C. (3) DNA extraction efficiency was significantly higher with the immediate thaw strategy than with gradient thawing at −20°C, −80°C, and in liquid nitrogen. (4) Among the conditions of −20°C, −80°C, and liquid nitrogen, −20°C was the best freezing condition for both improving DNA extraction efficiency and preserving microbial species diversity in meconium, while liquid nitrogen was the worst condition. (5) Three freeze-thaw cycles could markedly enhance DNA extraction efficiency and preserve the species diversity of meconium microflora. We developed a feasible freeze-thaw pretreatment protocol to improve the extraction of microbial DNA from meconium, which may be beneficial for newborn bacterial colonization studies.

Highlights

The human intestinal microflora is a highly diversified ecosystem composed of trillions of gastrointestinal bacteria with counts approximately 10 times the number of human cells (Bäckhed et al, 2005; Biagi et al, 2017)
The results showed that the DNA extraction efficiency of the frozen samples was generally higher than that of the control, and the efficiency at −20°C was higher than that at −80°C
The results showed that immediately cooling down to −80°C can result in a better improvement than gradient cooling (Figure 3B); immediate cooling in the liquid nitrogen condition showed no difference with gradient cooling in terms of the DNA extraction efficiency (Figure 3C), which implied that the temperature of freezing in liquid nitrogen was too low to damage the yield of DNA

Summary

Introduction

The human intestinal microflora is a highly diversified ecosystem composed of trillions of gastrointestinal bacteria with counts approximately 10 times the number of human cells (Bäckhed et al, 2005; Biagi et al, 2017). These microorganisms form the “human biome” by coexisting with the host (Sundman et al, 2017). In 2015 (Hansen et al, 2015), evidence demonstrated a low number of bacteria in first-pass meconium from healthy, vaginally delivered, breastfeeding infants. Human meconium contains complex microbial communities, and these bacteria may have and may continue to affect the development of the foetal immune system and host-microbe interactions

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Results

Conclusion