RT-PCR Used to Detect MERS Coronavirus in the Air of a Saudi Arabian Camel Barn

Saudi Arabian researchers have detected genetic fragments of Middle East Respiratory Syndrome coronavirus (MERS-CoV) in the air of a barn holding a camel infected with the virus. The work, published this week in mBio®, the online open-access journal of the American Society for Microbiology, indicates that further studies are needed to see if the disease can be transmitted through the air.

MERS, a serious viral respiratory illness caused by the coronavirus, has been identified in 699 people as of June 11, according to the World Health Organization; 209 people have died from the condition. An additional 113 cases occurring between 2012 and 2014 were reported by the Saudi Arabian Ministry of Health on June 3.

For the study, researchers on three consecutive days last November collected three air samples from a camel barn owned by a 43-year-old male MERS patient who lived south of the town of Jeddah, who later died from the condition. Four of the man's nine camels had shown signs of nasal discharge the week before the patient became ill; he had applied a topical medicine in the nose of one of the ill camels seven days before experiencing symptoms.

Using a laboratory technique called reverse transcription polymerase chain reaction (RT-PCR) to detect gene expression, they found that the first air sample, collected on November 7, contained genetic fragments of MERS-CoV. This was the same day that one of the patient's camels tested positive for the disease. The other samples did not test positive for MERS-CoV, suggesting short or intermittent shedding of the virus into the air surrounding the camels, said lead study author Esam Azhar, PhD, head of the Special Infectious Agents Unit at King Fahd Medical Research Center and associate professor of medical virology at King Abdulaziz University in Jeddah.

Additional experiments confirmed the presence of MERS-CoV-specific genetic sequences in the first air sample and found that these fragments were exactly identical to fragments detected in the camel and its sick owner.

"The clear message here is that detection of airborne MERS-CoV molecules, which were 100% identical with the viral genomic sequence detected from a camel actively shedding the virus in the same barn on the same day, warrants further investigations and measures to prevent possible airborne transmission of this deadly virus," Azhar said.

"This study also underscores the importance of obtaining a detailed clinical history with particular emphasis on any animal exposure for any MERS-CoV case, especially because recent reports suggest higher risk of MERS-CoV infections among people working with camels," he added.

Meanwhile, he said, mounting evidence for camel-to-human transmission of MERS-CoV warrants taking precautionary measures: People who care for camels or who work for slaughterhouses should wear face masks, gloves and protective clothing, and wash their hands frequently. It is also important to avoid contact with animals that are sick or have tested positive for MERS-CoV. Those who visit camel barns, farms or markets should wash hands before and after contact with animals. In addition, pasteurization of camel milk and proper cooking of camel meat are strongly recommended.

The study was supported by King Abdulaziz University.

Source: The American Society for Microbiology

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Nanopore Technique Rapidly Decodes Long DNA Strands

A low-cost technology may make it possible to read long sequences of DNA far more quickly than current techniques.

The research advances a technology, called nanopore DNA sequencing. If perfected it could someday be used to create handheld devices capable of quickly identifying DNA sequences from tissue samples and the environment, the University of Washington researchers who developed and tested the approach said.

“One reason why people are so excited about nanopore DNA sequencing is that the technology could possibly be used to create ‘tricorder’-like devices for detecting pathogens or diagnosing genetic disorders rapidly and on-the-spot,” said Andrew Laszlo, lead author and a graduate student in the laboratory of Jen Gundlach, a UW professor of physics who led the project.

The paper “Decoding long nanopore sequencing reads of natural DNA”  describes the new technique. It appears June 25 in the advanced online edition of the journal Nature Biotechnology.

Most of the current gene sequencing technologies require working with short snippets of DNA, typically 50 to 100 nucleotides long. These must be processed by large sequencers in a laboratory. The cumbersome process can take days to weeks to complete.

Nanopore technology takes advantage of the small, tunnel-like structures found in bacterial membranes. In nature, such pores allow bacteria to control the flow of nutrients across their membranes.

UW researcher used the nanopore Mycobacterium smegmatis porin A (MspA). This bacterial pore has been genetically altered so that the narrowest part of the channel has a diameter of about a nanometer, or 1 billionth of a meter. This is large enough for a single strand of DNA to pass through. The modified nanopore is then inserted into a membrane separating two salt solutions to create a channel connecting the two solutions.

To read a sequence of DNA with this system, a small voltage is applied across the membrane to make the ions of the salt solution flow through the nanopore. The ion flow creates a measurable current. If a strand of DNA is added to the solution on one side of the membrane and then enters a pore, the bulky DNA molecules will impede the flow of the much smaller ion and thereby alter the current. How much the current changes depends on which nucleotides — the individual molecules adenine, guanine, cytosine and thymine that make up the DNA chain — are inside the pore. Detecting changes in current can reveal which nucleotides are passing through the nanopore’s channel at any given instant.

Since the technique was first proposed in the 1990s, researchers hoped that nanopore DNA sequencing would offer a cheap, fast alternative to current gene sequencing. But their attempts have been frustrated by several challenges. It is difficult to identify each nucleotide one-by-one as they pass through the nanopore. Instead, researchers have to work with changes in current associated with four nucleotides at a time. In addition, some nucleotides may be missed or read more than once. Consequently, current nanopore sequencing technology yields an imprecise readout of a DNA sequence.

The UW researchers describe how they bypassed these problems. The researchers first identified the electronic signatures of all the nucleotide combinations possible with the four nucleotides that make up DNA — a total of 256 combinations in all (4 x 4 x 4 x 4).

They then created computer algorithms to match the current changes generated when a segment of DNA passes through the pore with current changes expected  from DNA sequences of known genes and genomes stored in a computer database. A match would show that the sequence of the DNA passing through the pore was identical or close to the DNA sequence stored in the database. The whole process would take minutes to a few hours, instead of weeks.

To test this approach, the researchers used their nanopore system to read the sequence of bacteriophage Phi X 174, a virus that infects bacteria and that is commonly used to evaluate new genome sequencing technologies. They found that the approach reliably read the bacteriophage’s DNA sequences and could  read sequences as long as 4,500 nucleotides.

“This is the first time anyone has shown that nanopores can be used to generate interpretable signatures corresponding to very long DNA sequences from real-world genomes,” said co-author Jay Shendure, a UW associate professor of genome sciences whose lab develops applications of genome sequencing technologies.  “It’s a major step forward.”

Because the technique relies on matching readings to databases of previously sequenced genes and genomes, it cannot yet be used to sequence a newly discovered gene or genome, the researchers said, but with some  refinements, they added, it should  be possible to improve performance in this area. To accelerate research on this new technology, the scientists are making their methods, data and computer algorithms fully available to all.

“Despite the remaining hurdles, our demonstration that a low-cost device can reliably read the sequences of naturally occurring DNA and can interpret DNA segments as long as 4,500 nucleotides in length represents a major advance in nanopore DNA sequencing,” Gundlach said.

This work was supported by the National Institutes of Health, National Human Genome Research Institutes $1,000 Genome Program Grants R01HG005115, R01HG006321 and R01HG006283  and a graduate research fellowship from the National Science Foundation DGE-0718124.

Source: University of Washington

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USP 1223 Now Available for Public Review and Comment

The USP Expert Microbiology Committee has recently published it's draft of USP 1223, Validation of Alternative Microbiological Methods.  Interested parties should visit the USP Pharmacopeial Forum (PF) website to review the draft.  The web address is http://www.usp.org/usp-nf/pharmacopeial-forum.  You are required to register to gain access to the USP PF online, but this is a free service to the pharmaceutical community. The public can provide comments on the draft chapter 1223 until September 30, 2014.  

This proposed revision provides guidance on the selection and implementation of assay methodologies to serve as alternatives to compendial microbiological methods. The revised chapter describes important steps that should be taken to evaluate candidate alternative methods, to select the analytical technology, and ultimately to qualify the method with actual product. These steps include, but are not limited to, identification of a potentially suitable alternative methodology, demonstration that the method is equivalent and applicable as a replacement for a standard compendial method, development of user specifications for equipment selection, and qualification of the method in the laboratory. In addition, this chapter outlines four distinct options for demonstrating equivalence.

A separate draft chapter, 1223.1, Validation of Alternative Methods to Antibiotic Microbial Assays, is also available for review and comment.

Microbiological assay methods are used to quantify the potency, or antimicrobial activity, of antibiotics. These microbial assays provide a direct measure of the effectiveness of the antibiotic against a reference microorganism. However, microbial assays have limited selectivity and are not appropriate for evaluating organic impurities. Also, the specific skill sets required for performing microbiological antibiotic assays, their unique equipment requirements, and their comparative complexity deter many stakeholders from using these methods. In contrast, physicochemical analytical procedures, such as high-performance liquid chromatography (HPLC), allow for simpler preparation and analysis of samples and rapid data acquisition with improved precision, accuracy, selectivity, and specificity. Alternative methods can be used effectively for both potency assignment and organic impurity testing. This proposed general chapter presents points to consider for stakeholders who wish to use physicochemical methods such as HPLC as alternatives to microbial assay methods described in general chapter Antibiotics—Microbial Assays 81.

The public can also provide comments on the draft 1223.1 until September 30, 2014. 

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