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Abstract

Introduction:

Safe handling of biological samples sourced from wild ecosystems is a pressing concern for scientists in disparate fields, including ecology and evolution, OneHealth initiatives, bioresources, geography, veterinary medicine, conservation, and many others. This is especially relevant given the growing global research community and collaborative networks that often span international borders. Treatments to inactivate potential pathogens of concern during transportation and analysis of biospecimens while preserving molecular structures of interest are necessary.

Objective:

We provide a detailed resource on the effectiveness and limitations of TRIzol™ Reagent, a product commonly used in molecular biology to inactivate bacterial and viral pathogens found in wild animals.

Methods:

By literature review, we evaluate the mode of action of TRIzol Reagent and its main components on bacterial and viral structures. We also synthesize peer-reviewed literature on the effectiveness of TRIzol in inactivating a broad range of infectious bacteria and viruses.

Key Findings:

TRIzol Reagent inactivation is based on phenol, chaotropic salts, and sodium acetate. We find evidence of widespread efficacy in deactivating bacteria and a broad range of enveloped viruses. The efficacy against a subset of potential pathogens, including some nonenveloped viruses, remains uncertain.

Conclusion:

Available evidence suggests that TRIzol Reagent is effective in inactivating a broad spectrum of bacteria and viruses from cells, tissues, and liquids in biological samples when the matrices are exposed to at least 10 min at room temperature to the reagent. We highlight areas that require additional research and discuss implications for laboratory protocols.

Introduction

Study of biological samples sourced from wild animals is essential for diverse research spanning ecology and evolution, biomedical health, and conservation, among others.^1–3 However, these samples may pose risks to the health and safety of human researchers, their networks, and the associated domesticated animals that provide essential foods and services. These concerns are especially relevant given the growing global research community and collaborative networks that often span international borders. Protocols for the safe storage, shipment, and analysis of specimens are required.

Many procedures aiming to inactivate potential pathogens rely on denaturation via exposure to high temperatures for extended periods.⁴ However, such protocols damage the molecules, including proteins, DNA, and RNA that may be the target of scientific investigation. As an alternative, identifying appropriate storage buffers that preserve molecular structures of interest while safely inactivating viruses and other pathogens of concern is essential. One potentially useful solution is TRIzol™ Reagent (Invitrogen™; hereafter, “TRIzol”), a popular agent for nucleic acid extractions that also has potent pathogen inactivation properties.^5–8 When samples are frozen soon after collection, the quantity and quality of DNA and RNA poststorage in TRIzol are suitable for many molecular applications.^9,10 Moreover, it has been demonstrated that long-term tissue storage (up to 9 years) in TRIzol does not observably impact RNA quality for gene expression assays.¹¹

Importantly, TRIzol has three constituents with properties that are known to inactivate bacterial and viral pathogens. However, a systematic review of the efficacy and limitations of TRIzol and of its individual constituents in inactivating pathogens is currently lacking.

In this study, we synthesize literature assessing the efficacy of TRIzol in inactivating viruses and bacteria. We provide available information on the composition of TRIzol and consider the impact of different constituents of this solvent on a diverse array of bacteria and viruses, including many that are representative of the pathogens potentially carried by wild animals. We additionally aim to highlight limitations in treatment efficacy or our knowledge or documentation of effects. Our goal is to provide a resource for researchers and other decision makers regarding the utility of TRIzol in inactivating pathogens of concern and to identify areas where future assessment is still needed.

Chemical and Antimicrobial/Virucidal Properties of TRIzol

TRIzol was developed by Chomczynski and Sacchi¹² in 1987 as an easy and fast method for RNA isolation. However, its utility for pathogen inactivation has increased its use in biological sample collection.^5–8 Chomczynski and Sacchi created a protein denaturing solution containing a mixture of 4 M guanidinium thiocyanate, 25 mM sodium citrate pH 7, 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol.¹² After adding 2 M sodium acetate pH 4, 1 mL phenol, and 0.2 mL chloroform-isoamyl alcohol mixture (49:1), RNA could be purified from the solution.¹²

Variations of this product are sold under various commercial names, including TRIzol Reagent, which is designed for solid samples, TRIzol LS Reagent, which is more concentrated and specifically designed for liquid samples, TRI Reagent™, TRIsure™, and TRIpure™ isolation reagent.^13,14 Although the exact composition of TRIzol Reagent (and the aforementioned related products) is proprietary and protected under U.S. Patent No. 5346994, information on its composition is available in material safety data sheets,¹⁵ as well as in published TRIzol-recreations.¹⁴

From these resources, it is documented that TRIzol contains the following: 30–60% (v/v) phenol (CAS-No 108-95-2), 0.8 M (15–40%) guanidine isothiocyanate (CAS-No 596-84-0), 0.4 M (7–13%) ammonium thiocyanate (CAS-No 1762-95-4), 0.1 M sodium acetate, and 5% (v/v) glycerol, along with other proprietary compounds, and has a pH of 3.^15,16 By reviewing the impact of each of the constituents on bacteria and viruses, an informed assessment of the inactivation potential of the reagent overall is possible.

The first TRIzol ingredient of importance, phenol (C₆H₅OH), also referred to as carbolic acid, is a flammable, corrosive, and toxic aromatic acid alcohol with bactericidal/virucidal properties (at 1–2%), meaning it has the ability to inactivate and thereby prevent the spread of microorganisms on various surfaces.^17–20 Furthermore, it is sporicidal at 5%,^19–22 but high temperatures might be required for this effect depending on the spore's nature.²² For decades, phenol and especially phenolic derivatives have been popular surface disinfectants in clinical settings and the food industry due to their ability to easily penetrate organic matter.^18,20 Phenols bind amino acid structures of membrane proteins, capsid proteins, and cytoplasmic enzymes causing denaturation or coagulation, and readily cause membrane damage and leakage of metabolites, which in turn interferes with the cell metabolism.^19,20,23

Through binding of fusion proteins, many phenolics are able to inhibit virus–host attachment and fusion.²⁴ Their high affinity to proteins is attributed to their hydrophobic benzenoid rings and hydroxyl groups. These properties have made phenol and phenolics widely recognized for being highly effective at inactivating bacteria, fungi, and some viruses, and preventing germination of highly resistant spores.^18,22,25,26 These same characteristics also confer important toxicity for the user, animals, and the environment; phenol is a hazardous substance, and safety protocols for its handling must be carefully followed.

The next TRIzol component, chaotropic salts, has strong protein dissociating properties. This is achieved via preferential binding of low-molecular-weight molecules and weakening of the hydrogen bonding networks of water molecules and amino acids, which increases protein solubility and is referred to as the Hofmeister effect.^27,28 Individual proteins dissociate to a variable degree due to the nature of their structural bonds (intermolecular hydrogen bonds, electrostatic and hydrophobic interactions) as well as additional contributing factors including pH, temperature, and concentration.²⁷ The extent to which chaotropic salts can destabilize proteins follows the Hofmeister ion series.²⁹ Both guanidinium cations (Gmd⁺) and thiocyanate anions (SCN⁻) are on the least hydrated side of this scale.^28,29 This weak hydration is a key aspect of the polypeptide and hydrophobic side chain binding mechanism that results in unfolding and exposing of amides, and increasing the solubility of nonpolar groups.²⁸

Guanidinium isothiocyanate (C₂H₆N₄S), the chaotropic salt found in TRIzol, is an extremely effective protein denaturant and has a well-documented ability to alter protein structures, resulting in loss of function across a wide diversity of applications.²⁸ In addition to its cell lysis function, guanidinium isothiocyanate is particularly efficient in breaking protein disulfide bonds of endogenous ribonucleases.^13,30 Ammonium thiocyanate (NH₄SCN), another TRIzol ingredient, is a weakly acidic chaotropic salt with well-established protein denaturing properties.³¹ The ammonium cation (NH₄⁺) has a high affinity to the carboxylate groups found on protein surfaces, which has been demonstrated to lead to protein clustering and precipitation.³²

Finally, the third component of TRIzol investigated regarding its pathogen inactivation properties is sodium acetate (C₂H₃NaO₂). This salt binds to the negatively charged polyphosphate backbone of nucleic acids and depolarizes the genomic material.³³ This allows DNA/RNA to precipitate when ethanol or isopropanol is added during the extraction protocol. In the food industry, sodium acetate has bacteriostatic and bactericidal properties.³⁴ The mechanism by which these effects are achieved is debated, however, it likely involves the cumulative effect of acidification of the cytoplasm and toxic accumulation of anions.^35–37 In the former, a process known as “energetic uncoupling,” acetic acid molecules enter the alkalic cell cytoplasm via passive diffusion where they dissociate into acetate anions and protons.^37,38 The cell expends energy to actively pump the protons out, consequently negatively affecting growth.

Another proposed mechanism involves the maintenance of osmotic pressure. To counteract the toxic accumulation of acetate anions, the cell expels them and reduces other intracellular anions involved in enzyme regulation, which has a direct impact on important metabolic pathways and growth.^35–38

Demonstrated Efficacy Against Bacteria

Structure and Inactivation of Bacteria

Bacteria are classified as gram-negative and gram-positive depending on their cellular structure. Gram-positive bacteria have a thick, often multilayered, protective peptidoglycan linked to other polymers, including teichoic acids, polysaccharides, and peptidoglycolipids.³⁹ The peptidoglycan mesh layer consists of alternating groups of N-acetylmuramic acid and N-acetylglucosamine linked by a β1,4 glycosidic bond. Certain antibiotics, such as penicillin, interfere with the peptidoglycan biosynthesis, and enzymes such as lysozyme can break the bond between the amino sugars. Gram-negative bacteria have a complex bilayer cell wall structure consisting of a much thinner peptidoglycan layer connected to a layer of complex lipopolysaccharides (LPS). The latter has endotoxic properties owing to the presence of lipid A and plays an important role in the pathogenesis of the bacterium.⁴⁰ Lipid A is the highly hydrophobic base structure of LPS, typically composed of a β−1′,6-linked disaccharide backbone carrying acyl chains (fatty acids) and two phosphoryl groups.^40,41

Various structural modifications to those acyl chains and phosphate groups result in variable endotoxic activity across gram-negative bacteria.^40,41 Between the bilayer and the cytoplasmic membrane, gram-negative bacteria have a periplasmic space that contains various proteins involved in membrane transport.

Due to their high affinity for proteins, disruption of the cell structure is the main mechanism by which phenols and chaotropic salts inactivate bacteria.^{18,20,42–47} Phenol causes a cascade of damaging events on the bacterial cell, including increased permeability of the cytoplasmic membrane causing leakage of intracellular metabolites and ions, binding and inactivating of membrane-embedded proteins, interference with the membrane fluidity and disrupting the cell's energy metabolism via passive proton flux, inhibition of ATP synthesis, and binding proteins involved in energy transduction (Table 1).^{18,20,44–48} Phenol at concentrations higher than 1% has tuberculocidal properties.²¹ In Escherichia coli, it was shown that ethanol and chaotropic salts weaken hydrophobic bonds of membrane wall and soluble proteins leading to the inhibition of peptidoglycan crosslinking and induction of cell death.⁴³ The efficacy of these chaotropic agents to promote cell lysis is linearly correlated with the Hofmeister series.

Table 1.

Summary of mechanisms of action of the three main TRIzol ingredients on bacterial and viral structures

	Phenol	Chaotropic salts	Sodium acetate
Bacteria	Disrupts cellular membranes by denaturing and inactivating integral and peripheral proteins and enzymes causing leakage of intracellular metabolites and ions^{18,20,44–47} Interferes with the membrane fluidity by introducing conformational changes in phospholipids and decreasing the lipid-to-protein ratio^47,48 Disrupts cellular energy metabolism via passive proton flux, inhibition of ATP synthesis, and binding proteins involved in energy transduction⁴⁶	Disrupts cellular membrane by weakening the hydrophobic bonds of membrane-bound and soluble proteins causing inhibition of the peptidoglycan crosslinking and destruction of internal cell structures^42,43 Inhibits cell wall synthesis and cell metabolism⁴³ Denatures and inactivates enzymes⁴²	Acidifies the cytoplasm causing energetic uncoupling and toxic accumulation of dissociated acid anions and protons, which leads to growth inhibition^35–37,49 Counteracts osmotic pressure via anion exchange, which directly impacts a cascade of metabolic reactions including enzyme activity, protein, DNA, and RNA synthesis, and cell growth³⁸ Inhibits the activity of intracellular enzymes and biosynthesis by altering the pH³⁵
Enveloped viruses	Inhibits virus–host attachment and fusion via (glyco)protein binding²⁴ Disrupts and destabilizes the viral envelope by solubilizing lipid bilayers and denaturing and precipitating proteins^20,23,76	Disrupts and destabilizes the viral envelope, solubilizes the lipid bilayers, and denatures proteins^23,65,66	Low pH (≤5) induces irreversible viral aggregation, leakage of the envelope, and conformational changes to structural proteins including glycoproteins^77,79 Acid inhibits enzymes and denatures proteins and nucleic acids⁷⁶ Organic acids interact with the lipophilic structures of the envelope⁷⁶
Nonenveloped viruses	Aggregates viral particles⁷³ Deforms the outer capsid⁷³ Forms fibrous material between viral particles⁷³	Aggregates viral particles⁷³ Denatures receptor proteins and blocks contact with host⁷³	No evidence found

Treatment of gram-positive bacteria with 4 M guanidine thiocyanate at 37°C for 5 h resulted in increased cell wall permeability, and destroyed the inner cell membranes and structures while leaving the nucleic acid molecules intact.⁴² Sodium acetate can diffuse across the cell membrane, thereby disrupting the bacteria's cytosolic pH homeostasis, which has a direct impact on a cascade of metabolic reactions including enzyme activity, protein, DNA, and RNA synthesis, and cell growth.^36,49

While effective against bacteria, phenolics and chaotropic salts have little tested effect against endospores produced by some groups including Bacillus and Clostridium, as endospores are some of the most durable structures found in nature. Although endospores are not usually a concern when dealing with biological samples, they present a potential challenge that should be evaluated if environmental matrices (water, soil) are studied. In this case, high temperatures can be highly effective as an inactivation method by damaging proteins involved in the endospore's metabolism.⁵⁰

Demonstrated Efficacy Against Viruses

Structure and Inactivation of Viruses

Viruses consist of a genomic (single- or double-stranded DNA or RNA) core surrounded by a cationic protein coat (capsid), also known as the nucleocapsid.^51,52 The capsid proteins are encoded by the viral genome and in many viruses they play a dual role in protecting the virus from the host immune response, as well as attachment and fusion with the host cell membrane.⁵³ In more complex viruses such as herpes, corona, and HIV, other structures are involved in providing protection against the host immune response and initiation of infection.⁵⁴ Viruses are classified into enveloped or nonenveloped viruses depending on the presence or absence of one or more additional lipid bilayers with proteins including glycoproteins.⁵² The lipid bilayer structure is derived from the host membranes and is mainly composed of cholesterol, followed by phospholipids and sphingolipids.⁵⁵

Examples of viral (glyco)proteins include envelope (E), membrane (M), and spike (S), as well as nonstructural proteins expressed by, for instance, dengue and chikungunya viruses or proteins produced for immune evasion such as semaphorins found in pox- and herpesviruses.⁵⁶ A fully infectious virus is referred to as a virion and its survival depends on the ability to deliver its genomic material into a host cell, where it is expressed.⁵¹ While they cannot replicate in the absence of a host cell, some DNA viruses such as Herpesviridae^57,58 and Parvoviridae⁵⁹ can remain dormant in the host and survive in the environment for long periods.⁶⁰

In contrast, many RNA viruses are capable of rapid acute infections in the susceptible host through various models of transmission.⁵⁸ In the absence of a susceptible host, these RNA viruses are easily eliminated. The structure of the capsid and envelope plays a key role in this transmission process and is a target for inactivation.⁵²

Inactivation of viruses generally involves inhibiting their life cycles by (1) altering the structure of the receptor, thus blocking contact with the host cell and infection, or (2) damaging the genomic material thus interfering with gene replication.⁵³ Potent virucidal agents are characterized by their ability to simultaneously target various structures. Sixty to 95% concentrated alcohols (e.g., ethanol, isopropyl alcohol) can inactivate viruses, but the efficacy is dependent on the surface properties of the microorganism.⁶¹ In general, the lipid bilayer of enveloped viruses is an easy target for lipid solvents including 80% ethanol in addition to acidic pH (<5) conditions.^61–64

Likewise, solutions containing chaotropic salts (such as guanidine thiocyanate, guanidine isothiocyanate) are extremely efficient in destabilizing viral envelopes by disrupting the capsid, solubilizing lipid bilayers, and denaturing proteins, thus effectively inactivating them (Table 1).^23,65,66 Chaotropic potency linearly correlates with the ability of chemical substances to induce structural and functional changes in enzymes and membranes with phenol (+143) and guanidine thiocyanate (+121) having a much stronger chaotropic potency than ethanol (+5.93).⁶⁷ In summary, enveloped viruses, which include members of Herpesviridae,^6,68 Togaviridae,^6,68 Poxviridae,⁶⁸ Flaviviridae,⁶ Bunyaviridae,⁶ and Coronaviridae,⁶⁹ along with many others inactivate quickly and effectively with chemical agents such as guanidine thiocyanate-based solutions and detergents.

Inactivation of nonenveloped viruses can be more challenging given their biochemical and biophysical characteristics.⁷⁰ Parvo-, noro-, and circoviruses are among the most resistant nonenveloped DNA viruses found in nature,⁷¹ yet it was demonstrated that the canine parvovirus, which serves as a model for human parvovirus B19, can be effectively inactivated after a 30-min exposure to 0.25% phenol (Table 2).⁷² In nonenveloped reoviruses, it was shown that treatment with 2.5 M guanidine-HCl or 1% phenol led to aggregation of viral particles.⁷³ Using electron microscopy, the authors revealed that the outer capsids of the virions became irregularly shaped and produced a large amount of fibrous material after treatment with phenol.

Table 2.

Inactivation by phenol

Family	Species	BSL	Genomic material	Viral titer, log₁₀ pfu/mL	Conc., %	Contact time, min	Temp.	Viral viability assay	Viral inactivation	Refs.
Enveloped
Herpesviridae	Bovine herpesvirus-1	2	dsDNA	NR	0.25	30	NR	TCID₅₀ assay, ELISA, CTA	≥8.11 log₁₀ reduction	⁷²
Flaviviridae	Bovine viral diarrhea virus	2	ssRNA(+)	NR	0.25	30	NR	TCID₅₀ assay, ELISA, CTA	≥7.58.11 log₁₀ reduction	⁷²
Paramyxoviridae	Canine distemper virus	2	ssRNA	NR	0.25	30	NR	TCID₅₀ assay, ELISA, CTA	≥8.12 log₁₀ reduction	⁷²
Rhabdoviridae	Vesicular stomatitis virus	2	ssRNA	NR	0.25	30	NR	TCID₅₀ assay, ELISA, CTA	≥8.99 log₁₀ reduction	⁷²
Coronaviridae	Canine coronavirus	2	ssRNA(+)	NR	0.25	30	NR	TCID₅₀ assay, ELISA, CTA	≥10.13 log₁₀ reduction	⁷²
Nonenveloped
Reoviridae	Bovine rotavirus	2	dsRNA	NR	0.25	30	NR	TCID₅₀ assay, ELISA, CTA	≥5.41 log₁₀ reduction	⁷²
Adenoviridae	Canine adenovirus-1	2	dsDNA	NR	0.25	30	NR	TCID₅₀ assay, ELISA, CTA	≥7.27 log₁₀ reduction	⁷²
Parvoviridae	Canine parvovirus	2	ssDNA	NR	0.25	30	NR	TCID₅₀ assay, ELISA, CTA	≥6.3 log₁₀ reduction	⁷²

BSL, biosafety level; CTA, cytotoxicity assay; ELISA, enzyme-linked immunosorbent assay; NR, not reported; TCID₅₀, median tissue culture infectious dose assay.

Guanidine-HCl, on the contrary, denatured the σ1 proteins of type 3 reovirus thus blocking host cell contact and preventing infection. Small positive-strand RNA viruses (+ssRNA viruses) such as poliovirus are especially difficult to inactivate as the genomic material may remain infectious.⁷⁴ When exposed to 6 M guanidine thiocyanate for 30 min, however, a greater than 4 log₁₀ reduction of the poliovirus infectivity can be achieved (Table 3).⁶⁸ While improper handling of RNA isolated from these viruses may impose an increased risk for infection, for many RNA viruses, successful transfection highly depends on a number of factors including introduction into a susceptible host reservoir and environmental conditions. Extreme acidic (pH <3) or alkaline (pH >12) conditions can result in denaturation of structural proteins and consequently interfere with the host interactions.⁷⁵

Table 3.

Inactivation by guanidine (iso)thiocyanate

Family	Species	BSL	Genomic material	Viral titer, log₁₀ pfu/mL	Conc., M	Contact time	Temp., °C	Viral viability assay	Viral inactivation	Refs.
Enveloped
Herpesviridae	Herpes simplex virus type I	2	dsDNA	NR	6	1	22	PA	>4.8 log₁₀ reduction	⁶⁸
Togaviridae	Sindbis	2	ssRNA(+)	NR	6	1	22	PA	4.9 log₁₀ reduction	⁶⁸
Poxviridae	Vaccinia	2	dsDNA	NR	6	1	22	PA	>5.4 log₁₀ reduction	⁶⁸
Nonenveloped
Picornaviridae	Polio 1	2	ssRNA(+)	NR	6	1	22	PA	4.1 log₁₀ reduction	⁶⁸

PA, plaque assay.

Besides viral structural features, humidity, pH, and temperature are also important to consider when evaluating inactivation methods, as lower temperatures, for instance, could lead to less effective inactivation.⁶¹ Furthermore, interactions between chemical and physical inactivators need to be taken into account. As an example, various strains of the avian influenza virus could be effectively inactivated under acidic conditions (pH <6) due to irreversible conformational changes to the spike protein involved in host–virion fusion.^76,77 However, in a saline-rich medium, the virions became more resistant to acidic conditions.⁷⁸ Moreover, the virus-inactivating properties of acidic pH decreased at lower temperatures (0°C). Similarly, exposure of HSV-1 to pH 5 for 3 h leads to irreversible changes in the fusion protein resulting in permanent inactivation.⁷⁹ Severe acute respiratory syndrome coronavirus (SARS-CoV)-1 is also completely inactivated under highly acidic conditions at 25°C and 37°C via altering the spike's protein binding ability.⁸⁰

Studies of TRIzol Inactivation of Pathogenic Bacteria and Viruses

We reviewed literature on the specific effectiveness of TRIzol Reagent, guanidinium (iso)thiocyanate, phenol, and acidic pH (<4) to inactivate bacteria and viruses associated with human and animal diseases (Tables 2–4). We searched PubMed and Google Scholar for the following key words: TRIzol, phenol(ics), guanidine (iso)thiocyanate, chaotropic agents, sodium acetate, inactivation, viruses, bacteria, pathogens. We identified 53 articles that met these search criteria and summarized the findings in the article and tables. In addition, we read the safety data sheets, standard operating procedures, and other technical manuals for TRIzol and its components to identify their characteristics. Finally, pathogen safety data sheets and biosafety manuals were searched for pathogen-specific information. Bacterial spores, Mycobacterium species, and small nonenveloped viruses generally show more resistance to chemical inactivation compared with medium-to-large-sized enveloped viruses.^4,61

Table 4.

TRIzol inactivation

Family	Species	BSL	Genomic material	Viral titer, log₁₀ pfu/mL	Virus: TRIzol ratio	Contact time, min	Temp.	Viral viability assay	Viral inactivation	Refs.
Enveloped
Togaviridae	Western equine encephalitis virus	3	ssRNA(+)	7.3	1:4	10	RT	PA	Complete	⁵
	Eastern equine encephalitis virus	3	ssRNA(+)	7	1:4	10	RT	PA	Complete	⁵
	Venezuelan equine encephalitis virus	3	ssRNA(+)	6.4	1:4	10	RT	IFA, CPE	Complete	⁷
				7.3	1:4	10	RT	PA	Complete	⁵
				11.6	1:3, 3:1 and 1:9	10	37°C	IFA	Complete	⁸
	Chikungunya virus	3	ssRNA(+)	7.3	1:4	10	RT	IFA, CPE	Complete	⁷
	Chikungunya virus	3	ssRNA(+)	7.7	1:10	10	RT	CPE	≥4.8 log₁₀ reduction, CPE negative	⁶
Flaviviridae	West Nile virus	3	ssRNA(+)	7	1:4	10	RT	PA	Complete	⁵
				7.07	1:9	10	NR	CPE, CTA	≥2.34 log₁₀ reduction, could still be detected and transfected	⁷⁰
				8.5	1:10	10	RT	CPE	≥4 log₁₀ reduction, CPE negative	⁶
	Zika virus	2	ssRNA(+)	8.81	1:3 and 9:1	48 h	NR	IFA	Complete	⁸
	Dengue virus	2	ssRNA(+)	5.47	1:4	10	RT	IFA, CPE	Complete	⁷
	Dengue virus	2	ssRNA(+)	4.9–8	1:4	10	RT	PA	Complete	⁵
Bunyaviridae	La Crosse virus	2	ssRNA	5.4	1:10	10	RT	CPE	≥1.7 log₁₀ reduction; CPE negative	⁶
	Rift Valley fever	3	ssRNA	7.21	1:4	10	RT	IFA, CPE	Complete	⁷
				6	1:4	10	RT	PA	Complete	⁵
				5.54 and 5.21	200 μL of clarified mosquito homogenate to 750 μL TRIzol LS™	NR	NR	CPE	Complete; CPE negative	⁶⁶
Orthomyxoviridae	Avian influenza virus	2	ssRNA	6.83–6.89	1:9	10	NR	CPE, CTA	≥2.39 and 2.45 log₁₀ reduction	⁷⁰
Orthomyxoviridae	Avian influenza virus	2	ssRNA	7	1:9	5–10	RT	CPE	Complete	⁸⁴
Paramyxoviridae	Nipah virus	4	ssRNA	6.61	1:4	10	RT	IFA, CPE	Complete	⁷
Coronaviridae	MERS-CoV	2	ssRNA(+)	6.28	1:4	10	RT	IFA, CPE	Complete	⁷
Coronaviridae	MERS-CoV	2	ssRNA(+)	9.5	1:3	10	RT	IFA, CPE	CPE negative	⁹³
	SARS-CoV-2	2	ssRNA(+)	NR	1:4	10	RT	dPCR	Reduction of viral RNA: N gene by 47.5% copies; ORF 1ab gene by 54.4%	⁹⁰
				5.7 (liquid virus), 6.7 (cells), and 8.7 (tissue)	1:2.2, 1:2, and 1:12	10	20°C	CPE	Complete, CPE negative	⁹²
				6	1:10	5–10	RT	CPE	Complete, CPE negative	⁹¹
				5.3	1:4	5	RT	CPE	Complete, CPE negative	⁸⁶
Arenaviridae	Guanarito virus	4	ssRNA	6.4	1:4	10	RT	IFA, CPE	Complete	⁷
	Sabia virus	4	ssRNA	6.38	1:4	10	RT	IFA, CPE	Complete	⁷
	Machupo	4	ssRNA	5.52	1:4	10	RT	IFA, CPE	Complete	⁷
	Lymphocytic choriomeningitis virus	3	ssRNA	5	1:4	10	RT	IFA, CPE	Complete	⁷
	Lujo hemorrhagic fever virus	4	ssRNA	5.35	1:4	10	RT	IFA, CPE	Complete	⁷
	Lassa virus	4	ssRNA	5.24	1:4	10	RT	IFA, CPE	Complete	⁷
Filoviridae	Marburg virus	4	ssRNA	6.61	1:4	10	RT	IFA, CPE	Complete	⁷
	Marburg virus	4	ssRNA	6.8–9	1:4	10	RT	PA	Complete	⁵
	Ebola virus	4	ssRNA	5.21–5.91	1:4	10	RT	IFA, CPE	Complete	⁷
				6	1:4	10	RT	PA	Complete	⁵
				5.7 (tissue)	1:4	10	RT	TCID₅₀	Complete, CPE negative	⁹⁸
Nonenveloped
Picornaviridae	Swine vesicular disease virus	3	ssRNA(+)	7.01–7.57	1:9	10	NR	CPE, CTA	≥2.84 and 2.28 log₁₀ reduction	⁷⁰

We report the log reduction values in the “viral inactivation” column when these data were provided by the referenced articles. In cases where this information was not provided, we use the term “complete” following the terminology used in the original publications.

CPE, cytopathic effect; IFA, immunofluorescence assay; MERS-CoV, Middle East respiratory syndrome coronavirus; RT, room temperature; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

In addition, the degree to which different bacteria and viruses react to TRIzol exposure depends on the taxon due to morphological variation.^81,82 The log titer reduction is generally accepted as the main parameter to measure the efficacy of the chemical agent and a 4 log₁₀ reduction can be translated to a 99.99% decrease of viral infectivity.⁶¹ Other key parameters include the contact time, the viral titer, and the concentration of the chemical. The Invitrogen manual recommends adding TRIzol Reagent in a 10:1 ratio to the samples for extracting RNA, while the TRIzol LS Reagent only requires a 3:1 ratio as it is more concentrated.⁸³

Exposing various animal tissues containing Francisella tularensis for 5 min to the TriPure isolation reagent (1:10 ratio) resulted in complete inactivation of the pathogen, that is, infectious agents were rendered inactive or are eliminated.⁸⁴ Specifically, samples of infected liver, lung, and spleen tissues, with a calculated bacterial burden of 690, 8500, and 4450 CFU/g, respectively, went from generating bacterial colonies that were “too numerous to count” in undiluted samples to 0 colonies in undiluted samples treated with a 10:1 ratio of TriPure to sample. Serial dilutions of the infected sample were also completely inactivated (0 bacterial colonies grown). It was also demonstrated that lysis buffers, such as TRIzol, containing high concentrations of guanidine-containing chaotropic salts made samples containing Mycobacterium tuberculosis, at concentrations as high as 10⁸ CFU/mL, nonviable.⁸⁵

Numerous studies have demonstrated the effectiveness of TRIzol to inactivate various infectious viruses (Table 4).^5–8,66,86 High titers (>10⁶ pfu/mL) of pathogens including nonenveloped swine vesicular disease virus, and enveloped equine encephalitis virus, chikungunya virus, Rift Valley fever virus, West Nile virus, Lassa virus, Marburg virus, and Ebola virus (EV) were all effectively inactivated following a 10-min incubation in TRIzol at 1:4 and 1:9 ratios (Table 2).^5–8,70 The effect of TRIzol was also tested on EV in tissue (liver) and cells of nonhuman primates showing virus inactivation in both cases using a 1:3 ratio after 5 min at room temperature.⁸⁷ This demonstrates that the efficacy of TRIzol to inactivate pathogens is not limited to isolated cells but can be applied to various matrices.⁸⁷

Hofmann et al.⁷⁴ also demonstrated the importance of the storage temperature with regard to RNA preservation in TRIzol, as none of the classical swine fever virus (CSFV) and only a few foot-and-mouth disease virus (FMDV) were able to replicate when stored at 37°C. The authors conclude that RNA purified from tissues containing CSFV or FMDV stored in TRIzol is not infectious unless directly transfected into susceptible cells. This observation is supported by the fact that the capsid of FMDV is extremely sensitive to acidic environments and, as a consequence, FMDV is readily inactivated when exposed to pH <6.^88,89

SARS-CoV-2, currently a global challenge, also had its exposure to TRIzol tested and its inactivation was effectively accomplished and recommended over heat treatment.^90–92 Another worldwide virus of concern of the same family, Middle East respiratory syndrome coronavirus (MERS-CoV), was also proved to be inactive in cells following a 10-min incubation at room temperature with TRIzol, allowing for further experimental work with MERS-CoV to be conducted at biosafety level 2 (BSL-2) biocontainment level.⁹³

While there are no studies published on the efficacy of TRIzol to inactivate members of the Herpesviridae family, HSV-1, which is genetically similar to the highly infectious Cercopithecine herpesvirus type 1 (herpes B),^63,94 showed a 4 log reduction after a 1-min exposure to 6 M guanidinium isothiocyanate (Table 3) as well as a 5-min exposure to acidic pH (<3).^62,68 In addition, Ngo et al.⁶ demonstrated that 7.0 log₁₀ pfu/mL of HSV-1 was inactivated when exposed to 500 μL easyMAG lysis buffer, which contains >50% guanidine thiocyanate, for 10 min at room temperature.⁹⁵ We acknowledge that TRIzol contains <6 M guanidinium thiocyanate, however, given that (1) chaotropic salts denature proteins and disrupt the capsid of enveloped viruses, (2) TRIzol contains multiple virucidal components including up to 60% phenol, and (3) TRIzol is acidic, similar results of inactivation can be expected for enveloped Herpesviridae.

Turning to the operational standards in North America, the standard operating protocols of virology laboratories (e.g., Provincial Laboratory for Public Health Alberta) routinely use DNA/RNA lysis reagents that contain guanidinium isothiocyanate, such as TRIzol (Dr. Kevin Fonseca, 2022, pers. comm.). These solutions are demonstrated to inactivate enveloped viruses and allow nucleic acid purification at a BSL-2 stage. In addition, Thermo Fisher Scientific confirmed that TRIzol will disrupt the viral envelope as well as protein function, and will solubilize lipids (Technical Applications Scientist, Life Sciences Solutions Group, 2022, pers. Comm.).

Importantly, official guidelines from a research institution outline the use of TRIzol for the inactivation of risk group-3 agents, and agree that this treatment after verification that the procedure inactivates the material in the user's laboratory according to the method's specifications allows transfer to BSL-2 laboratories.⁹⁶ Furthermore, pathogen safety data sheets from public institutions such as the Pathogen Regulation Directorate of the Public Health Agency of Canada (2011) describe infectious agents such as dengue virus susceptible to TRIzol.⁹⁷ This is evidence of the scientific trust that institutions have placed in the reagent as a safety resource for a laboratory setting when used according to verified procedures.

Limitations

Literature on TRIzol-based virus inactivation in clinical samples is not exhaustively assessed for all pathogens of concern, which is an important factor when evaluating its efficacy for the safe storage and handling of biological samples. However, many representative species and strains have been tested. One important note is that tissues or other organic structures (such as mucous) can form a protective layer around the pathogens and prevent inactivating agents from reaching the infectious organisms. This was demonstrated by storage of tissues (lymph node and tongue epithelium) containing FMDV and CSFV in TRIzol at various temperatures ranging from 0°C to 37°C.⁷⁴ While infectious RNA could still be isolated after 2 weeks of storage at all of the tested temperatures, the authors concluded that TRIzol greatly decreased risk of infectious RNA and that direct transfection would be required to propagate the virus into susceptible cells to cause infection.

In contrast, Haddock et al.⁹⁸ compared the efficacy of a number of different guanidinium isothiocyanate-based extraction buffers including TRIzol on liquid EV suspensions as well as infected tissues. Working in a BSL-4 laboratory, they demonstrated that when 50 mg of Ebola-infected tissue was incubated for 10 min in 1 mL TRIzol at room temperature, the virus was completely inactivated. Taken together, this indicates that in general, samples should be well homogenized in TRIzol for best efficacy. Furthermore, the majority of the reviewed literature involved testing the efficacy of TRIzol at room temperature. It is, however, known that the activity of chemical agents changes with temperature. For downstream molecular applications, it is recommended to preserve biological samples under frozen conditions (−20°C to −80°C).

Therefore, future studies should consider testing the efficacy of inactivating agents under different environmental conditions. In the meantime, holding samples at room temperature for at least 10 min could be done before freezing such that the expected results are obtained. While we review the mechanisms by which the different components of TRIzol act on bacterial cells, and we anticipate these effects to be generalizable, a comprehensive review of the effect of TRIzol on different pathogenic bacteria was not possible due to the limited literature available. In addition, other animal pathogens such as prions, Protozoa, and multicellular pathogenic parasites were not included in this review given the absence of information.

Including these pathogens in future research on the deactivation efficacy of TRIzol will further improve risk assessments. Assessments of the neutralization of viruses varied among studies, and may not have included measurements of cytotoxicity, which is a limitation that standardization of methods may resolve in future research. Finally, validation of the inactivation conditions for specific sample types is advisable and may be a legal requirement of select agent research. Region-specific laws should always be consulted and followed.

Conclusion

We reviewed the most relevant publications on the efficacy of TRIzol or its components to inactivate pathogens in biological samples. Three of the known constituents of TRIzol and their main mechanism of action for destroying bacteria and viruses are as follows: (1) phenol will disrupt the cell structure/viral envelope, and protein function; (2) chaotropic salts will promote protein dissociation; and (3) sodium acetate will promote cytoplasm acidification and depolarizes genomic material. The available data provide compelling evidence that each of these is effective in inactivating a broad spectrum of bacteria and viruses from cells, tissues, and liquids. In combination, the effects would be especially robust. All reviewed studies of TRIzol support the interpretation that TRIzol is effective as an inactivating agent for bacteria and enveloped viruses. No evidence to the contrary was present. More data are welcomed for nonenveloped viruses, which were assessed mainly indirectly by TRIzol constituents.

Available evidence suggests that the mechanisms discussed above will also inactivate nonenveloped viruses, especially if the matrices are exposed to TRIzol at a 1:9 ratio for at least 10 min at room temperature. TRIzol is attractive for scientific investigation due to its stabilizing properties that allow for long-term storage of samples and isolation of high-quality nucleic acids. In this study, we demonstrate that additional benefits include thorough inactivation of many pathogens of concern, especially bacteria and enveloped viruses, facilitating safe transport and handling of a diversity of biological samples. The published data on TRIzol provide valuable starting points for researchers planning future experiments exploring inactivation properties of agents of concern. Finally, it is important to emphasize that personnel handling potentially infectious materials should be wearing the appropriate personal protective equipment (gloves, laboratory safety glasses, working in biosafety cabinets, etc.) and handling the material commensurate with its risk level.

Footnotes

Acknowledgments

We would like to thank Drs. Douglas Morck, Steig Johnson, and Eoin O'Grady for valuable feedback on the earlier drafts of this article.

Authors' Contributions

G.D.: investigation (lead), methodology, and writing (original draft, review, and editing); A.D.M.: conceptualization, methodology, and writing (review and editing); P.R.S: investigation, methodology, and writing (review and editing); K.F. and F.v.d.M.: investigation and writing (review and editing).

Authors' Disclosure Statement

No competing financial interests exist.

Funding Information

The authors gratefully acknowledge support from the Natural Sciences and Engineering Research Council of Canada (NSERC-RGPIN/03782-2017, Amanda D. Melin), Canada Research Chairs program (CRC-2020-00015-Amanda D. Melin), and the University of Calgary (Amanda D. Melin, Gwen Duytschaever, and Patrícia R. Ströher).

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Effectiveness of TRIzol in Inactivating Animal Pathogens

Abstract

Introduction:

Objective:

Methods:

Key Findings:

Conclusion:

Introduction

Chemical and Antimicrobial/Virucidal Properties of TRIzol

Demonstrated Efficacy Against Bacteria

Structure and Inactivation of Bacteria

Demonstrated Efficacy Against Viruses

Structure and Inactivation of Viruses

Studies of TRIzol Inactivation of Pathogenic Bacteria and Viruses

Limitations

Conclusion

Footnotes

Acknowledgments

Authors' Contributions

Authors' Disclosure Statement

Funding Information

References