Detection of cytomegalovirus in the meconium of infected newborns by polymerase chain reaction

Villanueva, Maria Esterlita T; Svinarich, David M.; Gonik, Bernard; Ostrea, Enrique M.

doi:10.1002/1098-0997(2000)8:3/4<166::aid-idog12>3.0.co;2-x

Cited by 6 publications

(4 citation statements)

References 17 publications

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“…(2) The fragments of samples blocked the filter column, which affected the subsequent operations. (3) More PCR inhibitors ( Villanueva et al, 2000 ; Hansen et al, 2015 ) were loaded in the overloaded meconium. Interestingly, freeze-dried meconium showed a better DNA extraction efficiency than fresh meconium, possibly because freeze-dried meconium is homogeneous.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, the absolute content of microorganisms in meconium may also lead to a low DNA detection rate. Low levels of bacterial DNA are usually caused by high concentrations of PCR inhibitors in meconium ( Villanueva et al, 2000 ; Hansen et al, 2015 ), and the amplification rate of extracted DNA in meconium samples is commonly very low (10–52.9%; Ardissone et al, 2014 ; Hansen et al, 2015 ). Establishing a meconium-specific sampling extraction protocol is important for investigating the microbiome in the newborn gut ( Stinson et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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“…As a point of comparison, previously published work suggests that meconium yields 0.2 ± 0.4 ng of prokaryotic DNA per mg of meconium, compared with 16.6 ± 6.4 ng of prokaryotic DNA per mg of stool at 1 year of age (Wampach et al, 2017 ). The low yield of bacterial DNA from meconium is further complicated by its high concentrations of PCR inhibitors (Villanueva et al, 2000 ; Hansen et al, 2015 ). Meconium is a unique substance, and not stool in the traditional sense.…”

Section: Introductionmentioning

confidence: 99%

Comparison of Meconium DNA Extraction Methods for Use in Microbiome Studies

2018

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The establishment of human gut microbiota commences initially in utero. Meconium—the first fecal material passed after birth—can be used to study fetal gut contents; however, processing meconium samples for microbiome studies presents significant technical challenges. Meconium hosts a low biomass microbiome, is tar-like in texture and contains high concentrations of PCR inhibitors. This study aimed to evaluate four different DNA extraction methods to elucidate the most effective method for bacterial DNA recovery and sequencing analysis from first-pass meconium. Samples from five infants were collected and processed using the following extraction kits: (1) Qiagen QIAamp DNA Stool Mini (QS); (2) Qiagen QIAamp DNA Microbiome (QM); (3) MoBio PowerSoil (PS); (4) MoBio MagAttract PowerMicrobiome (PM). Additionally, Kit PM was employed with a double inhibitor removal treatment (IRT) step (PM2). Bacterial DNA recovery was assessed by qPCR. Any PCR inhibition in samples was measured by spiking DNA eluates with 0.1 ng of pure Streptococcus agalactiae (GBS) DNA followed by qPCR quantitation. Kit PM yielded the highest average total DNA yield (79.3 ng per gram of meconium). Samples extracted with kit PS had the highest detectable levels of 16S rRNA gene by qPCR. The ability of each kit to overcome PCR inhibition varied, with qPCR on GBS-spiked DNA from kits QS, QM, PS, and PM recovering 87.1, 91.0, 88.8, and 37.9% GBS DNA, respectively. Double IRT improved the performance of kit PM, increasing GBS recovery to 56.5%. However, once DNA yield was normalized to the level recovered with the other kits 100% of GBS DNA was detected, suggesting that levels of PCR inhibitors are related to DNA yield from kit PM. Ion Torrent 16S rRNA gene sequencing revealed a high level of inter-kit variation in meconium microbiome structure. In particular, kit QM showed a bias toward extracting Firmicute DNA, while the other kits extracted primarily Proteobacterial DNA. Choice of extraction kit greatly impacts on the ability to extract and detect bacterial DNA in meconium and on the microbiome community structure generated from these samples.

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“…Intrauterine transmission may be delayed several months, leading to false negative results; therefore, the time interval between maternal infection and prenatal diagnostic sampling is an important consideration (Revello, Sarasini, Zavattoni, Baldanti, & Gerna, 1998). New polymerase chain reaction-based methods for the detection of CMV in meconium of newborns (Villanueva, Svinarich, Gonik, & Ostrea, 2000) and in urine using ultrarapid real-time LightCycler polymerase chain reaction systems (Schalasta, Eggers, Schmid, & Enders, 2000) are being developed. Another method currently being investigated is the use of filter paper cards for the collection and analysis of neonatal blood samples for CMV screening (Barbi et al, 2000).…”

Section: Diagnosismentioning

confidence: 99%