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Comparison of PCR primer pairs in the detection of human rhinoviruses in nasopharyngeal aspirates
Journal of Virological Methods Journal of Virological
Methods
66 (1997)
139- 147
Comparison of PCR primer pairs in the detection of human rhinoviruses in nasopharyngeal aspirates Juhana
Santti *, Timo Hyypiti,
28 November
1996; received
Halonen
University of Turku. FIN-20520
Departtnent c!f Virology and Medic&y Research Laboratq: Received
Pekka
in revised form
14 March
1997; accepted
Turku. Finland
14 March
1997
Abstract
The development of a rapid and highly sensitive PCR assay for the detection of human rhinoviruses (HRVs) in nasopharyngeal aspirates is described. Two simple and fast commercial RNA extraction methods and four primer pairs were compared. The most sensitive RNA extraction method (Ultraspe@) and primer pair (A) were applied to detection of HRV RNA in 49 nasopharyngeal aspirates, of which 31 had previously been found culture-positive for HRVs. All culture-positive specimens were found positive by PCR. In addition, four of the 18 culture-negative samples were positive by PCR. Primer pair A, however, is not specific for rhinoviruses; it also amplifies enteroviruses, and thus an additional hybridization step with an HRV-specific probe is needed for group-specific diagnosis. The assay was able to detect an amount of HRV-1B RNA corresponding to 0.01 infected cells. In addition, about 50 ag (about 10 genomes) of purified HRV-1B and CBV-3 RNA still gave a signal with this primer pair. 0 1997 Elsevier Science B.V. Kr~~~~ru’s: PCR; Rhinovirus;
RNA extraction
1. Introduction Human rhinoviruses (HRVs), consisting of over 100 different serotypes, are the most frequent cause of the common cold. In addition, HRVs are increasingly recognized in lower respiratory tract
* Corresponding author. Tel.: + 358 21 3337033; 2 I 3337000; e-mail: [email protected] 0166.0934/97/$17.00
Q 1997 Elsevier
PII so I66-0934(97)00049-9
Science
fax: + ,358
B.V. All rights
reserved.
disease (Gwaltney, 1995). exacerbations of asthma and acute otitis media (Arola et al., 1988). The detection of HRVs has been carried out traditionally by culture of the virus followed by the acid lability test. The method is time-consuming and laborious, and it is not practised widely in diagnostic laboratories. Hence, faster and more sensitive detection systems would be highly appreciated and would improve our knowledge of the clinical importance of HRVs.
140
3. Suntti et al. /Journal of‘ Virological Methods 66 (1997) 139- 147
The detection of HRV RNA in clinical specimens by PCR has been described by several research groups (Gama et al., 1988, 1989; Hyypia et al., 1989; Torgersen et al., 1989; Bruce et al., 1990; Olive et al., 1990; Arruda and Hayden, 1993; Atmar and Georghiou, 1993; Ireland et al., 1993; Johnston et al., 1993; Kammerer et al., 1994; Mori and Clewley, 1994; Halonen et al., 1995; Arola et al., 1996). The oligonucleotide primers used in these studies were usually derived from highly conserved parts of 5’ non-coding (SNCR) and VP2 capsid protein-coding regions. Primers from the VP3 and the RNA polymerasecoding regions have also been used (Mori and Clewley, 1994). Some of these primer pairs have not distinguished directly between rhino- and enteroviruses (Gama et al., 1989; Torgersen et al., 1989; Bruce et al., 1990; Johnston et al., 1993; Kammerer et al., 1994; Mori and Clewley, 1994; Halonen et al., 1995) and final differentiation has been obtained by hybridization (Bruce et al., 1990; Johnston et al., 1993; Halonen et al., 1995) restriction enzyme analysis (Torgersen et al., 1989; Kammerer et al., 1994) or sequencing of amplicons (Mori and Clewley, 1994). The fact that rhinoviruses, unlike enteroviruses, have an apparent 120 base pair long deletion in the S’NCR has been taken advantage of by some investigators (Olive et al., 1990; Atmar and Georghiou, 1993; Ireland et al., 1993; Arola et al., 1996) in discrimination between amplicons originating from the two virus groups. The present study analyzed sequences of previously used primers and fourteen completely sequenced human picornavirus genomes. On the basis of study data, we selected four primer pairs and assessed their efficacy for direct detection of HRVs in clinical specimens.
2. Materials
and methods
2.1. Clinical specimens The specimens included 49 nasopharyngeal aspirates from the specimen collection of the Department of Virology, University of Turku. They were originally sent to the diagnostic unit for
respiratory virus isolation and antigen detection (Halonen et al., 1985). Of the 49 specimens, 31 were rhinovirus culture positive and one was also positive for cytomegalovirus (Arola et al., 1988). Ten of the rhinovirus negative specimens were positive for adenovirus. The specimens had been stored at - 40°C for several years. 2.2. RNA
extraction
2.2.1. Proteinuse K-SDS-Phenol extraction Fifty ~1 of the samples were digested with 500 ~1 of proteinase K-SDS solution (at concentrations of 100 pug/ml and 0.5%, respectively) at 37°C for 1 h and phenol extracted twice: After addition of 1 ml of phenol, brief vortexing and centrifugation at 12 000 x g for 3 min, the upper aqueous phases were carefully transferred to new tubes; 200 ~1 of chloroform was added, the tubes were briefly vortexed, centrifuged at 12 000 x g for 2 min, and the upper phases were collected. The nucleic acids were precipitated with 1 vol of absolute ethanol in the presence of 0.3 M sodium acetate overnight at - 20°C pelleted by 30 min centrifugation at 12 000 x g (4°C) and the pellets were washed with 500 ,ul of cold 70% ethanol. The ethanol was removed, the pellets were air dried, dissolved in 50 ~1 of distilled water, and placed on ice before subsequent cDNA reactions. 2.2.2. Guanidium isothiocyanate extraction Two commercial RNA extraction reagents, Ultraspec’w and Ultraspec 3’B, were used according to the manufacturer’s (Cinna/Biotecx Laboratories Inc., Houston, TX, USA) instructions with the modifications. In the Ultraspec’” following method, 100 ~1 of the original samples was added to tubes containing 500 ~1 of carefully mixed Ultraspec” reagent. The mixtures were briefly vortexed and cooled on ice for 5 min to permit complete dissociation of virus particles. One hundred ~1 chloroform was added, the tubes were vortexed for 30 s, cooled on ice for 5 min, and centrifuged at 12 000 x g (4°C) for 15 min. Most of the upper phase (350 ~1) containing RNA (while DNA and proteins occur in the lower organic phase) was transferred to tubes containing 350 ~1 isopropanol and briefly vortexed. The
J. Sanrri et al. 1Journal
of’ 1‘irolugicrrl hluthoci.s 66 (1997)
samples were stored for 1 h at - 20°C for precipitation and centrifuged at 12 000 x g (4°C) for 20 min. The pellets were then washed once with 600 !!I of 75% cold ethanol, air dried, dissolved in 50 ![I of distilled water, incubated at 55°C for 15 min, and finally placed on ice before cDNA reactions. In the Ultraspec 3’ method, 100 ~1 of the samples was mixed with 1 ml of the reagent, and 200 ,ll of chloroform was added. After 30 s of vortexing and centrifugation at 12000 x g (4Y‘) for 15 min, 400 ,~ll of the upper phase was transferred to tubes containing 200 ~1 isopropanol and vortexed. Twenty ~11 RNA Tack” resin was added, the mixtures were vortexed for 30 s and centrifuged at 12 000 x g (4°C) for 1 min. The pellets were washed once with 1 ml of 75% cold ethanol, air dried and dissolved in 50 ~1 of distilled water. After incubation at 55°C for 15 min and centrifugation at 7000 x g for 30 s, supernatants were collected in fresh tubes and placed on ice. 2.3. Oligonuchtidt~
prinwrs
The oligonucleotide primers were derived from the highly conserved parts of the 5’ non-coding and VP2 capsid protein-coding regions of the picornavirus genome. Both previously described (e.g. Gama et al., 1988, 1989; Hyypii et al., 1989; Haloncn et al., 1995) and newly modified conserved and more specific primers were used. Fig. 1 shows the location of the primers. Their length varied from 16 to 2 1 nucleotides and the sizes of the amplicons with HRV-2 were 120 (pair A), 204 (B), 96 (C) and 533 (D) base pairs. Fig. 2 shows the alignment of primer sequences with selected rhinovirus and enterovirus serotypes.
< II Fig. I. Location genome.
m of primer
pairs
A, B, C and
D in HRV-2
2.4. Recrrse
139
147
trunscription
141
(RT)
RT mixture with a total volume of 40 /tl containing 22.2 ~1 of RNA extract, 50 mM Tris-HCl (pH 8.3), 75 mM KC], 3 mM MgCl,, 10 mM DTT, 0.5 mM dNTPs (Pharmacia, Uppsala, Sweden), 4 U rRNasin”’ ribonuclease inhibitor (Promega, Madison, WI), 20 U of M-MLV reverse transcriptase (Promega) and 50 pmol of one of the antisense ( - ) primers was prepared. The reaction mixtures were incubated at 37°C for 1 h. 2.5. PCR Ten ~1 of cDNA reaction product was added to the PCR reaction mixture with a total volume of 100 111. The mixture contained 10 mM Tris-HCl (pH 8.8). 50 mM KC]. 1.5 mM MgCI,, 0.1% Triton X-100, 0.2 mM dNTPs, 1 U thermostable DNA polymerase (Dynazyme”; Finnzymes, Espoo, Finland) and 50 pmol of one of the sense ( + ) and antisense ( - ) primers. Two drops of mineral oil was added into the tubes to prevent vapor formation. Amplification was carried out in a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) with the following, previously described (Halonen et al., 1995) program: 3 min denaturation at 94°C followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 45 s and extension at 72°C for 1 min. After the last cycle, 7 min extension at 72°C was carried out. Another program for primer pair D resulting in longer amplicons was used with 40 cycles of denaturation at 95°C for 2 min, annealing at 55°C for 2 min, and extension at 72°C for 4 min. Twenty ~11 of the PCR products was electrophoresed in 2% agarose gels containing 10 /lg/rnl of ethidium bromide in 0.5 x Tris/borate/ EDTA buffer (TBE, pH 8.0). The amplicons were visualized under ultraviolet light. To prevent contamination, the tests were carried out in different rooms and under different hoods, including (i) RNA extraction, (ii) preparation of RT and PCR reaction mixtures, (iii) pipetting of RNA extracts and cDNA reaction products, and (iv) gel electrophoresis. The pipette tips used were aerosol resistant. The gloves were changed frequently and the tubes were opened
J. Santti et al. /Journal
142
PRIMER PAIR
3+ HRV-1B HRV-2
A
of’ Virological Methods 66 (1997) 139-147
HRV-14 HRV-89 PV- 1 PV-2 PV-3 CBV-1 CBV-3 CSV-4 CBV-5 CAV-9
CAV-16 CAV-21 QQXOMIC! IX)C!ATION
5'TccTccQQccccMMTo ____---___________ --________-----___ __________---_____ __________________ __________________ -_________---_____ __________-_-_____ __________________ ____-_____________ -_________-------___-_-____________ --________------__ -_________--_--___ ________-_________
3'
-____----- ______---__ _____--- -_______-____ __-- _--______________ _-- _________ -- _______ _____----- -_____---__ _____-----_____ _---__ _____--_________- --__
____________ - ________ _____---- _______ - --__
434-451
533-553
RSPE+ HRV-1B HRV-2 mtv-14 Hrlv-89
B
PV-1 PV-2 PV-3 CBV-1 CBV-3 cm-4 CBV-5 CM-9 CAV-16 CAV-21 -C
4-
5 ’ AGCCMcoMocMcC __________---_-_ _____----_---___ ____----____&__ __--____________ __&_____T_~_~_ __G____-_T__Q___ --~---__CT-~-~T-O-_____T-w-‘+ T_C(______T-w-G_ T-&_____T-W-QT_&_____T-w_& ~&_-_-CT-O__& T-&-e-_-T-w-& __&_____T_~_&
3' _____________-_______ _________-_---_______ _____------_____----_____----_-_____----_____________________ ____________--_______ _________-_----______ _____-----_-____-----__-____________-_--_ _____________________ _____________________
533-553
350-365
LOCATION
FtsPE2-
3+
n
L
HRV-18 HRV-2 Imv-14 ERV-89 PV-1 PV-2 PV- 3 CBV-1 CBV-3 CBV-4 CBV-5 cm-9 CAV-16 CAV-21
5'TccTccQQccccToMM __________--______ -_________-------__________------___________________ __________-------__-_-----_-_______ --_---_--_________ _-_--_-___________ _-__-_____________ __________________ _-________---------____________--_________-_-_______ ____-___-__-______
3'TTMcQcccTP.cccTQQ 5'MTrQcQQQAYQQQAcc _________________
3
5' 3'
___-C____________ -_(J______________ _-&Cm-T-&__A_-__O_T--T-‘3-e-A_-__cJ_C--T-&__A____‘._~-~&__A_-__C_~-~~---j,_-__C_~-~&--A_-__C_~-~&__A_-__C_~-~&__A___-C-CT-~&_-A_-_-&CT-T-&--A_-513-529
434-451 iiCATION
5-
4+ '3'
5'TACTTMOOT6CC
D
Iiuv-1e m-2 Imv-14 HRV-89 PV-1 PV-2 PV-3 CBV-1 CBV-3 CEW-4 CBV-5 CAV-9 CAV-16 CAV-21 GBkDMIC
LOCATION
_________ _-----_ _____ __________
_ ----_____
___________
_
--
________
- _--_________ ----
_
_ _
_
_
_
_ _
_
_
_
_
_
_
_
_
_
_
_
_________--___u____
_____________
__C______________A__
---___
_
-_T-_____________A__ __A____________C----
-------__~~~~~~~~~--~ _ _ _ _ _ _ _ _ _ _ - - - - - _ _ _ _ _ _
-__-________-_-__C__
-------
__________
-_A-_
_______
- __---________
---_
____
_-_--AL--C-e
-____________--__C__
----______-------____
--A-___~_____A_-Cm_
_--_______---------__
-_A_________-_-C-AL-_
533-553
::
____A_-A--
_-C-____~_____AL--A_-
-
-_
__________
______
__&_--__--___A_____
-----_
___________________ ______
__A_
-_&__________~~:_A--
_____________________
----
3p!cAccAccAcc p lTcNca
1046-1065
Fig. 2. Alignment and genomic location (HRV-2) of primer sequences (genomic polarity) used in the study with representatives rhino- and enteroviruses. Nucleotides differing from the primer are indicated. R is either A or G.
of
J. Santti rt al. /Journal Table Results
143
of Sirological Methods 66 (1997) 13% 147
I
Specimen
of a comparison number
of two RNA
extraction
Reagent
Ultraspec Ultraspec Ultraspec Ultraspec Ultraspec Ultraspec Ultraspec Ultraspec
methods, Dilution
3 3 3 3
Ultraspec”
and Ultraspec
3”, tested with four clinical
of specimen
IO-’
IO-”
+++ +++ +++ +++ ++ + +++ +++
++
IO
’
IO J
_
_ _
+++ ++ _ _
++ + _ _
++ _ _ _
+++ +++
+++ ++
++ _
A series of IO-fold dilutions made from RNA extracts were reverse transcribed and amplified agarose gel electrophoresis. Grading of bands: clearly visible (+ + +), visible (+ +), lightly visible (+). and not visible
with small pieces of paper. Two tubes were never kept open at the same time. Negative controls (distilled water) were included in every step.
3. Results 3.1. Compurison
of RNA
extraction
specimens
methods
In a preliminary study, the conventional proteinase K-phenol-chloroform extraction method was compared with the Ultraspecm method using nasopharyngeal aspirates. The methods were found to be equally sensitive, but owing to the fact that the proteinase K-phenolchloroform method is more time-consuming and laborious than the Ultraspecs protocols the latter were selected for further comparison. Four nasopharyngeal aspirates, positive for HRV isolation, were selected for the comparison of the two commercial quanidium isothiocyanate RNA extraction methods (Ultraspec”’ and Ultraspec 3’9, which are basically similar, the difference being in the isopropanol precipitation step. In the latter method, resin binds the RNA and no isopropanol precipitation is required. Both methods are relatively fast: ten samples can be processed in less than 3.5 h by Ultraspec@ and in less than 2.5 h by Ultraspec 3”. RNA was extracted from the four samples simultaneously with both methods. From each
using primer
IO i
I(1 ”
_
pair A and analysed
in
(- ).
extracted sample, a series of IO-fold dilutions (10 ~ ’ to 10 ~ ‘) in distilled water was produced. They were subsequently reverse transcribed and amplified with primer pair A. After agarose gel electrophoresis, specific amplicons from Ultraspec” treated samples were detected in about 10 times higher dilutions than those from IJltraspec 3”‘-treated samples (Table 1). In all further experiments Ultraspec’ was used for RNA extraction. 3.2. Comparison
of primers
Two nasopharyngeal aspirates positive for HRV isolation were selected to determine the most sensitive primer pair. After RNA extraction, lo-fold dilutions from 10 ’ to 10 ’ were produced, RNA was reverse transcribed and cDNA amplified in parallel using different primer pairs (A, B, C and D). Agarose gel electrophoresis showed visible bands in dilutions of 10 ’ (samples 1 and 2) with primer pair A, 10 ’ and 10 ’ with B, 10 ~~’ with C and no visible band with D, respectively (Table 2). The most sensitive primer pair, A, was used in all the subsequent tests. In a preliminary study, a primer pair with RSPE + as a sense primer and 3 - as an antisense primer was constructed, but it yielded nonspecific double bands, although the primers showed perfect complementarity with the serotypes tested. Raising of the annealing temperature to 60°C did not eliminate the extra band.
J. Suntti et al. i Journal of’ Virologid
144 Table 2 Results of a comparison Primer
pair
A B C D
of four primer Sample
pairs (A, B. C and D) tested Dilution
66 (1997) 139- 147
with two clinical
specimens
of specimen
10-l
IO 1
1 2 I
+++ +++ +++
+++ +++ ++
+ ++
2
+++
+++
++
_
_
I
+++
+++
__
._.
_
2
+++
+++
_
_
_
_
._
I
After RNA extraction, a series of IO-fold dilutions Grading of bands from agarose gel electrophoresis:
pair was therefore
_ _ _
_
2
The primer study.
Methd
excluded
_ _ _
_ _
were made, reverse transcribed, and amplified using different primer pairs. clearly visible (+ + + ), visible (+ + ). lightly visible (+), and not visible (-).
from the
3.3. Sensitivity of assay To determine the sensitivity of the test, 10” HeLa (Ohio) cells were infected with HRV-1B. When most of the cells showed typical CPE, they were collected, 500 ~1 of Ultraspec? was added, and RNA extraction was carried out as described. A series of lo-fold dilutions in distilled water were reverse transcribed and amplified using primer pair A. After agarose gel electrophoresis, a specific amplicon was detected in a lo-’ dilution of the RNA extract correponding to 0.01 infected cells. In addition, about 50 ag (about 10 genomes) of purified HRV-IB and CBV-3 RNA still gave a signal with this primer pair. 3.4. Analysis of selected entero- und rhinovirus sero types For evaluation of the specificity of the four primer pairs, selected enteroand rhinovirus serotypes (prototype strains, ATCC) were tested. Primer pair A resulted in a specific amplification product for all the tested HRV and enterovirus serotypes (HRV-lB, -2, -9, -11, -12, -14, -22, -89, CAV-16, -21, CBV-3 and PV-3) as expected. Primer pair B amplified all the HRV serotypes tested (HRV-2, -9, -14, -22, -89) and, unexpectedly, also enteroviruses (CAV-16, -21, CBV-3 and
PV-3). All HRV serotypes tested (HRV-2, -9, - 11, -14, -22, -89) were positive with primer pair C, which exhibited no reactivity with the enterovirus serotypes tested (CAV-16, -21, CBV-3, PV-3). Primer pair D resulted in a specific 650 bp amplicon for PV-3, CBV-3 and CAV-16, in a 630 bp amplicon for CAV-21 and in a 530 bp amplicon for HRV-2, -9, -22 and -89. In the case of HRV14, the electrophoretic analysis showed a rather prominent non-specific band and only a faint band of expected size (clearly seen after overloading of the gel; data not shown). In addition, only a faint, though visible band resulted for HRV-9. The fact that CAV-21 gave a slightly shorter amplicon than the other enteroviruses was an expected result of the sequence analysis and is seen in the representative results for primer pairs A and D in Fig. 3. The results of the analysis of selected entero- and rhinovirus serotypes are summarised in Table 3.
3.5. Cornpurison of PCR und virus culture with nusopharyngeal uspirates Using the most sensitive primer pair (A), all 31 culture-positive specimens were positive in PCR, and of the 18 culture-negative samples, four were PCR positive. Of these four HRV isolation negative but PCR positive samples, three were positive for adenovirus isolation.
J. Sanrri
et al. /Journal
qf’ C’irological
primer pair A
1.W
147
145
primer pair D II
I
M~~ihoris 66 (1997)
1
Fig. 3. Electrophoretic analysis of PCR products obtained with nine selected rhino- and enterovirus serotypes using primer pairs A and D in ethidium bromide stained 2% agarose gel. A DNA molecular size marker is in the first and last lanes. CONTROL is a negative control (distilled water). Arrows show the 120 bp amplicon obtained with primer pair A and the 530 bp (HRVs) and 650 bp (enteroviruses) amplicons obtained with primer pair D. Expectedly, this primer pair resulted in a slightly smaller amplicon (630 bp) for C‘AV-21 than for the other enteroviruses. HRV-I4 failed to yield a visible band of expected size with this primer pair.
4. Discussion The current method of HRV of the virus followed by acid laborious and time-consuming. of HRVs using this method is
Table 3 Results of PCR of selected usingthe four primer pairs Virus
HRV-2 HVR-Y HVR-I4 HVR-22 HVR-89 CAV-I6 CAV-21 CBV-3 PV-3
Primer
entero-
detection, culture lability testing, is Routine diagnosis of limited clinical
and
rhinovirus
serotype
pair
A
B
C
D
+ + + + + + + + +
+ + + + + + + + +
+ + + + + _ _ _ _
+ ,’ +” t’l + ,’ + “ +h +’ +h +b
,’ Smaller product, 530 bp. h Larger product, 650 bp. ’ Intermediate product, 630 bp.
value since it does not provide a specific diagnosis in the acute phase of illness. In this study we were able to single out a rapid and sensitive RT-PCR test for HRVs with potential to replace virus isolation in HRV diagnosis. We selected nasopharyngeal aspirates for the comparisons to find direct evidence for the most sensitive RNA extraction method and primer pair for samples of this type. The best extraction method and primer pair were also evaluated with infected cells and purified virus to determine the sensitivity in molecular terms. However, these data cannot be directly applied to clinical specimens, which may contain additional substances affecting results. The conventional proteinase K-SDS-phenol method with several phenol/chloroform extractions was found laborious and time-consuming for efficient routine diagnosis of HRVs. as observed previously (Arruda and Hayden, 1993). The two commercial RNA extraction methods compared here were found simple and fast, which is of high value when multiple samples are processed simultaneously. The Ultraspec’” method was about ten times more sensitive than the Ultra-
146
J. Suntti et al. /Journal
qf’ Virological Methods 66 (1997) 139-147
spec 3” method. Moreover, it took less than 3.5 h to treat ten samples with Ultraspec@. With some further modifications it may be possible to shorten the time required for RNA extraction even more. For example, we used 60 min for isopropanol precipitation. This time could evidently be reduced to 10 min as recommended by the manufacturer. The results indicate that of the four primer pairs tested, A was the most sensitive. All culturepositive specimens were positive with this primer pair, and, in addition, four of the 18 culture-negative specimens were PCR positive. Using the Ultraspec” procedure and primer pair A, we specifically detected an amount of the target corresponding to 0.01 HRV-1B infected cells. About 50 ag (about 10 genomes) of purified HRV-1 B and CBV-3 RNA still gave a signal with this primer pair. One disadvantage of primer pair A is, however, that it does not distinguish between rhino- and enteroviruses. This problem could be overcome by using HRV-specific probes for hybridization as described previously (Halonen et al., 1995). On the other hand, primer pair C could be used, particularly when HRV etiology is suspected in some unusual syndromes, although it is less sensitive. Using primer pair A there should be few if any false negatives, since the primers show perfect complementarity with selected rhino- and enterovirus serotypes as Fig. 2 shows. The fact that primer pair D showed lower sensitivity and resulted in a non-specific amplification product with HRV-14 may be due to the mismatches found with the antisense primer (5 - ) in different virus serotypes (Fig. 2). The poor sensitivity may also be partly due to the large size of the amplicon obtained with this primer pair. It may also be assumed that the repeated freezethaw cycles of the specimens in the comparison of primer pairs would lead to the degradation of HRV RNA. This could primarily decrease the sensitivity of primer pair D, which has a relatively long target RNA sequence compared with the other primer pairs studied here. In general, the low sensitivity of some of the primers used could depend on the rhinovirus type in the sample. The reason why selected enterovirus serotypes (CAV-16, -21, CBV-3 and PV-3) were positive, i.e.
they resulted in about 200 bp amplicon with primer pair B, remains to be determined. Since the antisense primer (4 - ) of this pair does not distinguish between rhino- and enteroviruses, the problem must be in the HRV-specific sense primer (RSPE +). In fact, regions in the enterovirus genome close to the primer site exhibit complementarity of five nucleotides with the 3’ end of the primer, and this may be sufficient for amplification when cell culture materials containing high quantities of HRV RNA are used. In conclusion, we describe optimization of a rapid and sensitive RT-PCR test for HRVs, which is superior to the current culture method in sensitivity and rapidity and could eventually replace the latter in the routine diagnosis of HRVs. Our test can be completed during one working day and thus provides a specific diagnosis in the acute phase of the illness, which is important for the prevention of unnecessary use of antibiotics, particularly in lower respiratory tract infections. On the other hand, rapid diagnostic assays may become important for the optimal use of candidate anti-rhinoviral drugs in the future. Owing to the better sensitivity of this test, the possible role of HRVs in various conditions other than the common cold could also be explored further.
Acknowledgements
We thank Anita Arola and Olli Ruuskanen for critical comments, Marita Maaronen for excellent technical assistance and Mikko Arola for clinical samples. This study was supported by grants from the Academy of Finland and the Sigrid Juselius Foundation.
References Arola, A., Santti, J., Ruuskanen, O., Halonen, P., Hyypia, T., 1996. Identification of enteroviruses in clinical specimens by competitive PCR followed by genetic typing using sequence analysis. J. Clin. Microbial. 34, 3133318. Arola, M.. Ziegler, T., Ruuskanen, O., Mertsola, .I., NantoSalonen, K., Halonen, P., 1988. Rhinoviruses in acute otitis media. J. Pediatr. 113, 693-695.
J. Suntti et ul. /Journal of Virological Methods 66 (1997) 139 147 Arruda, E., Hayden, F.G., 1993. Detection of human rhinovirus RNA in nasal washings by PCR. Mol. Cell. Probes 7. 373 --379. Atmar, R.L., Georghiou. P.R., 1993. Classification of respiratory tract picornavirus isolates as enteroviruses or rhinoviruses by using reverse transcription-polymerase chain reaction. J. Clin. Microbial. 31. 2544-2546. Bruce, C.B.. Gama, R.E., Hughes, P.J., Stanway, G., 1990. A novel method of typing rhinoviruses using the product of a polymerase chain reaction. Arch. Virol. 113, 83-87. Gama, R.E., Hughes, P.J., Bruce, C.B., Stanway. G., 1984. Polymerase chain reaction amplification of rhinovirus nucleic acids from clinical material. Nucleic Acids Res. 16, 9346. Gama. R.E.. Horsnell, P.R., Hughes, P.J., North, C.. Bruce, C.B.. Al-Nakib, W., Stanway. G., 1989. Amplification of rhinovirus specific nucleic acids from clinical samples using the polymerase chain reaction. J. Med. Virol. 28, 73- 77. Gwaltney, J.M. Jr., 1995. Rhinovirus. In: Mandell, B., Bennett. D.. Dolin, M., (Eds.), Principles and Practice of Infectious Diseases. Churchill Livingstone, New York. pp. 16% 1663. Halonen. P.. Obert, G., Hierholzer, J.C., 1985. Direct detection of viral antigens in respiratory infections by immunoassays: A four-year experience and new developments. In: de la Maza, L.M., Peterson, E.M.. (Eds.), Medical Virology IV. Lawrence Erlbaum, New Jersey. pp. 65-83. Halonen. P.. Rocha. E., Hierholzer, J., Holloway, B., Hyypiii.
141
T., Hurskainen, P., Pallansch. M., 1995. Detection 01 enteroviruses and rhinoviruses in clinical specimens by PCR and liquid-phase hybridization. J. Clin. Microbial. 33, 648 653. HyypiH. T., Auvinen, P.. Maaronen. M., 1989. Polymerasc chain reaction for human picornaviruses. J. Gen. Virol. 70. 3261. 3268. Ireland, D.C.. Kent, J.. Nicholson, K.G.. 1993. Improved detection of rhinoviruses in nasal and throat swabs b! seminested RT-PCR. J. Med. Virol. 40. 96 101. Johnston. S.L.. Sanderson, G.. Patternore. P.K.. Smith, S.. Bardin, P.G., Bruce, C.B., Lambden, P.R., Tyrrell, D.A.J.. Holgate, S.T.. 1993. Use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms. J. Clin. Microbial. 3 I. I I I 117. KHmmerer, U.. Kunkel, B., Korn. K., 1904. Nested PCR for specific detection and rapid identification of human picornaviruses. J. Clin. Microbial. 32, 285 291. Mori, J., Clewley. J.P., 1994. Polymerase chain reaction and sequencing for typing rhinovirus RNA. J. Med. Virol. 44. 323-329. Olive, D.M.. Al-Mufti. S., Al-Mulla. W.. Khan. M.A.. Pasta, A., Stanway. G., Al-Nakib. W., 1990. Detection and differentiation of picornaviruses in clinical samples following genomic amplification. J. Gen. Virol. 71, 2141 :!147. Torgersen, H., Skern. T., Blaas, D.. 1989. Typing of human rhinoviruses based on sequence variations in the 5’ noncoding region. J. Gen. Virol. 70. 31 I I 31 16.
Methods
66 (1997)
139- 147
Comparison of PCR primer pairs in the detection of human rhinoviruses in nasopharyngeal aspirates Juhana
Santti *, Timo Hyypiti,
28 November
1996; received
Halonen
University of Turku. FIN-20520
Departtnent c!f Virology and Medic&y Research Laboratq: Received
Pekka
in revised form
14 March
1997; accepted
Turku. Finland
14 March
1997
Abstract
The development of a rapid and highly sensitive PCR assay for the detection of human rhinoviruses (HRVs) in nasopharyngeal aspirates is described. Two simple and fast commercial RNA extraction methods and four primer pairs were compared. The most sensitive RNA extraction method (Ultraspe@) and primer pair (A) were applied to detection of HRV RNA in 49 nasopharyngeal aspirates, of which 31 had previously been found culture-positive for HRVs. All culture-positive specimens were found positive by PCR. In addition, four of the 18 culture-negative samples were positive by PCR. Primer pair A, however, is not specific for rhinoviruses; it also amplifies enteroviruses, and thus an additional hybridization step with an HRV-specific probe is needed for group-specific diagnosis. The assay was able to detect an amount of HRV-1B RNA corresponding to 0.01 infected cells. In addition, about 50 ag (about 10 genomes) of purified HRV-1B and CBV-3 RNA still gave a signal with this primer pair. 0 1997 Elsevier Science B.V. Kr~~~~ru’s: PCR; Rhinovirus;
RNA extraction
1. Introduction Human rhinoviruses (HRVs), consisting of over 100 different serotypes, are the most frequent cause of the common cold. In addition, HRVs are increasingly recognized in lower respiratory tract
* Corresponding author. Tel.: + 358 21 3337033; 2 I 3337000; e-mail: [email protected] 0166.0934/97/$17.00
Q 1997 Elsevier
PII so I66-0934(97)00049-9
Science
fax: + ,358
B.V. All rights
reserved.
disease (Gwaltney, 1995). exacerbations of asthma and acute otitis media (Arola et al., 1988). The detection of HRVs has been carried out traditionally by culture of the virus followed by the acid lability test. The method is time-consuming and laborious, and it is not practised widely in diagnostic laboratories. Hence, faster and more sensitive detection systems would be highly appreciated and would improve our knowledge of the clinical importance of HRVs.
140
3. Suntti et al. /Journal of‘ Virological Methods 66 (1997) 139- 147
The detection of HRV RNA in clinical specimens by PCR has been described by several research groups (Gama et al., 1988, 1989; Hyypia et al., 1989; Torgersen et al., 1989; Bruce et al., 1990; Olive et al., 1990; Arruda and Hayden, 1993; Atmar and Georghiou, 1993; Ireland et al., 1993; Johnston et al., 1993; Kammerer et al., 1994; Mori and Clewley, 1994; Halonen et al., 1995; Arola et al., 1996). The oligonucleotide primers used in these studies were usually derived from highly conserved parts of 5’ non-coding (SNCR) and VP2 capsid protein-coding regions. Primers from the VP3 and the RNA polymerasecoding regions have also been used (Mori and Clewley, 1994). Some of these primer pairs have not distinguished directly between rhino- and enteroviruses (Gama et al., 1989; Torgersen et al., 1989; Bruce et al., 1990; Johnston et al., 1993; Kammerer et al., 1994; Mori and Clewley, 1994; Halonen et al., 1995) and final differentiation has been obtained by hybridization (Bruce et al., 1990; Johnston et al., 1993; Halonen et al., 1995) restriction enzyme analysis (Torgersen et al., 1989; Kammerer et al., 1994) or sequencing of amplicons (Mori and Clewley, 1994). The fact that rhinoviruses, unlike enteroviruses, have an apparent 120 base pair long deletion in the S’NCR has been taken advantage of by some investigators (Olive et al., 1990; Atmar and Georghiou, 1993; Ireland et al., 1993; Arola et al., 1996) in discrimination between amplicons originating from the two virus groups. The present study analyzed sequences of previously used primers and fourteen completely sequenced human picornavirus genomes. On the basis of study data, we selected four primer pairs and assessed their efficacy for direct detection of HRVs in clinical specimens.
2. Materials
and methods
2.1. Clinical specimens The specimens included 49 nasopharyngeal aspirates from the specimen collection of the Department of Virology, University of Turku. They were originally sent to the diagnostic unit for
respiratory virus isolation and antigen detection (Halonen et al., 1985). Of the 49 specimens, 31 were rhinovirus culture positive and one was also positive for cytomegalovirus (Arola et al., 1988). Ten of the rhinovirus negative specimens were positive for adenovirus. The specimens had been stored at - 40°C for several years. 2.2. RNA
extraction
2.2.1. Proteinuse K-SDS-Phenol extraction Fifty ~1 of the samples were digested with 500 ~1 of proteinase K-SDS solution (at concentrations of 100 pug/ml and 0.5%, respectively) at 37°C for 1 h and phenol extracted twice: After addition of 1 ml of phenol, brief vortexing and centrifugation at 12 000 x g for 3 min, the upper aqueous phases were carefully transferred to new tubes; 200 ~1 of chloroform was added, the tubes were briefly vortexed, centrifuged at 12 000 x g for 2 min, and the upper phases were collected. The nucleic acids were precipitated with 1 vol of absolute ethanol in the presence of 0.3 M sodium acetate overnight at - 20°C pelleted by 30 min centrifugation at 12 000 x g (4°C) and the pellets were washed with 500 ,ul of cold 70% ethanol. The ethanol was removed, the pellets were air dried, dissolved in 50 ~1 of distilled water, and placed on ice before subsequent cDNA reactions. 2.2.2. Guanidium isothiocyanate extraction Two commercial RNA extraction reagents, Ultraspec’w and Ultraspec 3’B, were used according to the manufacturer’s (Cinna/Biotecx Laboratories Inc., Houston, TX, USA) instructions with the modifications. In the Ultraspec’” following method, 100 ~1 of the original samples was added to tubes containing 500 ~1 of carefully mixed Ultraspec” reagent. The mixtures were briefly vortexed and cooled on ice for 5 min to permit complete dissociation of virus particles. One hundred ~1 chloroform was added, the tubes were vortexed for 30 s, cooled on ice for 5 min, and centrifuged at 12 000 x g (4°C) for 15 min. Most of the upper phase (350 ~1) containing RNA (while DNA and proteins occur in the lower organic phase) was transferred to tubes containing 350 ~1 isopropanol and briefly vortexed. The
J. Sanrri et al. 1Journal
of’ 1‘irolugicrrl hluthoci.s 66 (1997)
samples were stored for 1 h at - 20°C for precipitation and centrifuged at 12 000 x g (4°C) for 20 min. The pellets were then washed once with 600 !!I of 75% cold ethanol, air dried, dissolved in 50 ![I of distilled water, incubated at 55°C for 15 min, and finally placed on ice before cDNA reactions. In the Ultraspec 3’ method, 100 ~1 of the samples was mixed with 1 ml of the reagent, and 200 ,ll of chloroform was added. After 30 s of vortexing and centrifugation at 12000 x g (4Y‘) for 15 min, 400 ,~ll of the upper phase was transferred to tubes containing 200 ~1 isopropanol and vortexed. Twenty ~11 RNA Tack” resin was added, the mixtures were vortexed for 30 s and centrifuged at 12 000 x g (4°C) for 1 min. The pellets were washed once with 1 ml of 75% cold ethanol, air dried and dissolved in 50 ~1 of distilled water. After incubation at 55°C for 15 min and centrifugation at 7000 x g for 30 s, supernatants were collected in fresh tubes and placed on ice. 2.3. Oligonuchtidt~
prinwrs
The oligonucleotide primers were derived from the highly conserved parts of the 5’ non-coding and VP2 capsid protein-coding regions of the picornavirus genome. Both previously described (e.g. Gama et al., 1988, 1989; Hyypii et al., 1989; Haloncn et al., 1995) and newly modified conserved and more specific primers were used. Fig. 1 shows the location of the primers. Their length varied from 16 to 2 1 nucleotides and the sizes of the amplicons with HRV-2 were 120 (pair A), 204 (B), 96 (C) and 533 (D) base pairs. Fig. 2 shows the alignment of primer sequences with selected rhinovirus and enterovirus serotypes.
< II Fig. I. Location genome.
m of primer
pairs
A, B, C and
D in HRV-2
2.4. Recrrse
139
147
trunscription
141
(RT)
RT mixture with a total volume of 40 /tl containing 22.2 ~1 of RNA extract, 50 mM Tris-HCl (pH 8.3), 75 mM KC], 3 mM MgCl,, 10 mM DTT, 0.5 mM dNTPs (Pharmacia, Uppsala, Sweden), 4 U rRNasin”’ ribonuclease inhibitor (Promega, Madison, WI), 20 U of M-MLV reverse transcriptase (Promega) and 50 pmol of one of the antisense ( - ) primers was prepared. The reaction mixtures were incubated at 37°C for 1 h. 2.5. PCR Ten ~1 of cDNA reaction product was added to the PCR reaction mixture with a total volume of 100 111. The mixture contained 10 mM Tris-HCl (pH 8.8). 50 mM KC]. 1.5 mM MgCI,, 0.1% Triton X-100, 0.2 mM dNTPs, 1 U thermostable DNA polymerase (Dynazyme”; Finnzymes, Espoo, Finland) and 50 pmol of one of the sense ( + ) and antisense ( - ) primers. Two drops of mineral oil was added into the tubes to prevent vapor formation. Amplification was carried out in a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) with the following, previously described (Halonen et al., 1995) program: 3 min denaturation at 94°C followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 45 s and extension at 72°C for 1 min. After the last cycle, 7 min extension at 72°C was carried out. Another program for primer pair D resulting in longer amplicons was used with 40 cycles of denaturation at 95°C for 2 min, annealing at 55°C for 2 min, and extension at 72°C for 4 min. Twenty ~11 of the PCR products was electrophoresed in 2% agarose gels containing 10 /lg/rnl of ethidium bromide in 0.5 x Tris/borate/ EDTA buffer (TBE, pH 8.0). The amplicons were visualized under ultraviolet light. To prevent contamination, the tests were carried out in different rooms and under different hoods, including (i) RNA extraction, (ii) preparation of RT and PCR reaction mixtures, (iii) pipetting of RNA extracts and cDNA reaction products, and (iv) gel electrophoresis. The pipette tips used were aerosol resistant. The gloves were changed frequently and the tubes were opened
J. Santti et al. /Journal
142
PRIMER PAIR
3+ HRV-1B HRV-2
A
of’ Virological Methods 66 (1997) 139-147
HRV-14 HRV-89 PV- 1 PV-2 PV-3 CBV-1 CBV-3 CSV-4 CBV-5 CAV-9
CAV-16 CAV-21 QQXOMIC! IX)C!ATION
5'TccTccQQccccMMTo ____---___________ --________-----___ __________---_____ __________________ __________________ -_________---_____ __________-_-_____ __________________ ____-_____________ -_________-------___-_-____________ --________------__ -_________--_--___ ________-_________
3'
-____----- ______---__ _____--- -_______-____ __-- _--______________ _-- _________ -- _______ _____----- -_____---__ _____-----_____ _---__ _____--_________- --__
____________ - ________ _____---- _______ - --__
434-451
533-553
RSPE+ HRV-1B HRV-2 mtv-14 Hrlv-89
B
PV-1 PV-2 PV-3 CBV-1 CBV-3 cm-4 CBV-5 CM-9 CAV-16 CAV-21 -C
4-
5 ’ AGCCMcoMocMcC __________---_-_ _____----_---___ ____----____&__ __--____________ __&_____T_~_~_ __G____-_T__Q___ --~---__CT-~-~T-O-_____T-w-‘+ T_C(______T-w-G_ T-&_____T-W-QT_&_____T-w_& ~&_-_-CT-O__& T-&-e-_-T-w-& __&_____T_~_&
3' _____________-_______ _________-_---_______ _____------_____----_____----_-_____----_____________________ ____________--_______ _________-_----______ _____-----_-____-----__-____________-_--_ _____________________ _____________________
533-553
350-365
LOCATION
FtsPE2-
3+
n
L
HRV-18 HRV-2 Imv-14 ERV-89 PV-1 PV-2 PV- 3 CBV-1 CBV-3 CBV-4 CBV-5 cm-9 CAV-16 CAV-21
5'TccTccQQccccToMM __________--______ -_________-------__________------___________________ __________-------__-_-----_-_______ --_---_--_________ _-_--_-___________ _-__-_____________ __________________ _-________---------____________--_________-_-_______ ____-___-__-______
3'TTMcQcccTP.cccTQQ 5'MTrQcQQQAYQQQAcc _________________
3
5' 3'
___-C____________ -_(J______________ _-&Cm-T-&__A_-__O_T--T-‘3-e-A_-__cJ_C--T-&__A____‘._~-~&__A_-__C_~-~~---j,_-__C_~-~&--A_-__C_~-~&__A_-__C_~-~&__A___-C-CT-~&_-A_-_-&CT-T-&--A_-513-529
434-451 iiCATION
5-
4+ '3'
5'TACTTMOOT6CC
D
Iiuv-1e m-2 Imv-14 HRV-89 PV-1 PV-2 PV-3 CBV-1 CBV-3 CEW-4 CBV-5 CAV-9 CAV-16 CAV-21 GBkDMIC
LOCATION
_________ _-----_ _____ __________
_ ----_____
___________
_
--
________
- _--_________ ----
_
_ _
_
_
_
_ _
_
_
_
_
_
_
_
_
_
_
_
_________--___u____
_____________
__C______________A__
---___
_
-_T-_____________A__ __A____________C----
-------__~~~~~~~~~--~ _ _ _ _ _ _ _ _ _ _ - - - - - _ _ _ _ _ _
-__-________-_-__C__
-------
__________
-_A-_
_______
- __---________
---_
____
_-_--AL--C-e
-____________--__C__
----______-------____
--A-___~_____A_-Cm_
_--_______---------__
-_A_________-_-C-AL-_
533-553
::
____A_-A--
_-C-____~_____AL--A_-
-
-_
__________
______
__&_--__--___A_____
-----_
___________________ ______
__A_
-_&__________~~:_A--
_____________________
----
3p!cAccAccAcc p lTcNca
1046-1065
Fig. 2. Alignment and genomic location (HRV-2) of primer sequences (genomic polarity) used in the study with representatives rhino- and enteroviruses. Nucleotides differing from the primer are indicated. R is either A or G.
of
J. Santti rt al. /Journal Table Results
143
of Sirological Methods 66 (1997) 13% 147
I
Specimen
of a comparison number
of two RNA
extraction
Reagent
Ultraspec Ultraspec Ultraspec Ultraspec Ultraspec Ultraspec Ultraspec Ultraspec
methods, Dilution
3 3 3 3
Ultraspec”
and Ultraspec
3”, tested with four clinical
of specimen
IO-’
IO-”
+++ +++ +++ +++ ++ + +++ +++
++
IO
’
IO J
_
_ _
+++ ++ _ _
++ + _ _
++ _ _ _
+++ +++
+++ ++
++ _
A series of IO-fold dilutions made from RNA extracts were reverse transcribed and amplified agarose gel electrophoresis. Grading of bands: clearly visible (+ + +), visible (+ +), lightly visible (+). and not visible
with small pieces of paper. Two tubes were never kept open at the same time. Negative controls (distilled water) were included in every step.
3. Results 3.1. Compurison
of RNA
extraction
specimens
methods
In a preliminary study, the conventional proteinase K-phenol-chloroform extraction method was compared with the Ultraspecm method using nasopharyngeal aspirates. The methods were found to be equally sensitive, but owing to the fact that the proteinase K-phenolchloroform method is more time-consuming and laborious than the Ultraspecs protocols the latter were selected for further comparison. Four nasopharyngeal aspirates, positive for HRV isolation, were selected for the comparison of the two commercial quanidium isothiocyanate RNA extraction methods (Ultraspec”’ and Ultraspec 3’9, which are basically similar, the difference being in the isopropanol precipitation step. In the latter method, resin binds the RNA and no isopropanol precipitation is required. Both methods are relatively fast: ten samples can be processed in less than 3.5 h by Ultraspec@ and in less than 2.5 h by Ultraspec 3”. RNA was extracted from the four samples simultaneously with both methods. From each
using primer
IO i
I(1 ”
_
pair A and analysed
in
(- ).
extracted sample, a series of IO-fold dilutions (10 ~ ’ to 10 ~ ‘) in distilled water was produced. They were subsequently reverse transcribed and amplified with primer pair A. After agarose gel electrophoresis, specific amplicons from Ultraspec” treated samples were detected in about 10 times higher dilutions than those from IJltraspec 3”‘-treated samples (Table 1). In all further experiments Ultraspec’ was used for RNA extraction. 3.2. Comparison
of primers
Two nasopharyngeal aspirates positive for HRV isolation were selected to determine the most sensitive primer pair. After RNA extraction, lo-fold dilutions from 10 ’ to 10 ’ were produced, RNA was reverse transcribed and cDNA amplified in parallel using different primer pairs (A, B, C and D). Agarose gel electrophoresis showed visible bands in dilutions of 10 ’ (samples 1 and 2) with primer pair A, 10 ’ and 10 ’ with B, 10 ~~’ with C and no visible band with D, respectively (Table 2). The most sensitive primer pair, A, was used in all the subsequent tests. In a preliminary study, a primer pair with RSPE + as a sense primer and 3 - as an antisense primer was constructed, but it yielded nonspecific double bands, although the primers showed perfect complementarity with the serotypes tested. Raising of the annealing temperature to 60°C did not eliminate the extra band.
J. Suntti et al. i Journal of’ Virologid
144 Table 2 Results of a comparison Primer
pair
A B C D
of four primer Sample
pairs (A, B. C and D) tested Dilution
66 (1997) 139- 147
with two clinical
specimens
of specimen
10-l
IO 1
1 2 I
+++ +++ +++
+++ +++ ++
+ ++
2
+++
+++
++
_
_
I
+++
+++
__
._.
_
2
+++
+++
_
_
_
_
._
I
After RNA extraction, a series of IO-fold dilutions Grading of bands from agarose gel electrophoresis:
pair was therefore
_ _ _
_
2
The primer study.
Methd
excluded
_ _ _
_ _
were made, reverse transcribed, and amplified using different primer pairs. clearly visible (+ + + ), visible (+ + ). lightly visible (+), and not visible (-).
from the
3.3. Sensitivity of assay To determine the sensitivity of the test, 10” HeLa (Ohio) cells were infected with HRV-1B. When most of the cells showed typical CPE, they were collected, 500 ~1 of Ultraspec? was added, and RNA extraction was carried out as described. A series of lo-fold dilutions in distilled water were reverse transcribed and amplified using primer pair A. After agarose gel electrophoresis, a specific amplicon was detected in a lo-’ dilution of the RNA extract correponding to 0.01 infected cells. In addition, about 50 ag (about 10 genomes) of purified HRV-IB and CBV-3 RNA still gave a signal with this primer pair. 3.4. Analysis of selected entero- und rhinovirus sero types For evaluation of the specificity of the four primer pairs, selected enteroand rhinovirus serotypes (prototype strains, ATCC) were tested. Primer pair A resulted in a specific amplification product for all the tested HRV and enterovirus serotypes (HRV-lB, -2, -9, -11, -12, -14, -22, -89, CAV-16, -21, CBV-3 and PV-3) as expected. Primer pair B amplified all the HRV serotypes tested (HRV-2, -9, -14, -22, -89) and, unexpectedly, also enteroviruses (CAV-16, -21, CBV-3 and
PV-3). All HRV serotypes tested (HRV-2, -9, - 11, -14, -22, -89) were positive with primer pair C, which exhibited no reactivity with the enterovirus serotypes tested (CAV-16, -21, CBV-3, PV-3). Primer pair D resulted in a specific 650 bp amplicon for PV-3, CBV-3 and CAV-16, in a 630 bp amplicon for CAV-21 and in a 530 bp amplicon for HRV-2, -9, -22 and -89. In the case of HRV14, the electrophoretic analysis showed a rather prominent non-specific band and only a faint band of expected size (clearly seen after overloading of the gel; data not shown). In addition, only a faint, though visible band resulted for HRV-9. The fact that CAV-21 gave a slightly shorter amplicon than the other enteroviruses was an expected result of the sequence analysis and is seen in the representative results for primer pairs A and D in Fig. 3. The results of the analysis of selected entero- and rhinovirus serotypes are summarised in Table 3.
3.5. Cornpurison of PCR und virus culture with nusopharyngeal uspirates Using the most sensitive primer pair (A), all 31 culture-positive specimens were positive in PCR, and of the 18 culture-negative samples, four were PCR positive. Of these four HRV isolation negative but PCR positive samples, three were positive for adenovirus isolation.
J. Sanrri
et al. /Journal
qf’ C’irological
primer pair A
1.W
147
145
primer pair D II
I
M~~ihoris 66 (1997)
1
Fig. 3. Electrophoretic analysis of PCR products obtained with nine selected rhino- and enterovirus serotypes using primer pairs A and D in ethidium bromide stained 2% agarose gel. A DNA molecular size marker is in the first and last lanes. CONTROL is a negative control (distilled water). Arrows show the 120 bp amplicon obtained with primer pair A and the 530 bp (HRVs) and 650 bp (enteroviruses) amplicons obtained with primer pair D. Expectedly, this primer pair resulted in a slightly smaller amplicon (630 bp) for C‘AV-21 than for the other enteroviruses. HRV-I4 failed to yield a visible band of expected size with this primer pair.
4. Discussion The current method of HRV of the virus followed by acid laborious and time-consuming. of HRVs using this method is
Table 3 Results of PCR of selected usingthe four primer pairs Virus
HRV-2 HVR-Y HVR-I4 HVR-22 HVR-89 CAV-I6 CAV-21 CBV-3 PV-3
Primer
entero-
detection, culture lability testing, is Routine diagnosis of limited clinical
and
rhinovirus
serotype
pair
A
B
C
D
+ + + + + + + + +
+ + + + + + + + +
+ + + + + _ _ _ _
+ ,’ +” t’l + ,’ + “ +h +’ +h +b
,’ Smaller product, 530 bp. h Larger product, 650 bp. ’ Intermediate product, 630 bp.
value since it does not provide a specific diagnosis in the acute phase of illness. In this study we were able to single out a rapid and sensitive RT-PCR test for HRVs with potential to replace virus isolation in HRV diagnosis. We selected nasopharyngeal aspirates for the comparisons to find direct evidence for the most sensitive RNA extraction method and primer pair for samples of this type. The best extraction method and primer pair were also evaluated with infected cells and purified virus to determine the sensitivity in molecular terms. However, these data cannot be directly applied to clinical specimens, which may contain additional substances affecting results. The conventional proteinase K-SDS-phenol method with several phenol/chloroform extractions was found laborious and time-consuming for efficient routine diagnosis of HRVs. as observed previously (Arruda and Hayden, 1993). The two commercial RNA extraction methods compared here were found simple and fast, which is of high value when multiple samples are processed simultaneously. The Ultraspec’” method was about ten times more sensitive than the Ultra-
146
J. Suntti et al. /Journal
qf’ Virological Methods 66 (1997) 139-147
spec 3” method. Moreover, it took less than 3.5 h to treat ten samples with Ultraspec@. With some further modifications it may be possible to shorten the time required for RNA extraction even more. For example, we used 60 min for isopropanol precipitation. This time could evidently be reduced to 10 min as recommended by the manufacturer. The results indicate that of the four primer pairs tested, A was the most sensitive. All culturepositive specimens were positive with this primer pair, and, in addition, four of the 18 culture-negative specimens were PCR positive. Using the Ultraspec” procedure and primer pair A, we specifically detected an amount of the target corresponding to 0.01 HRV-1B infected cells. About 50 ag (about 10 genomes) of purified HRV-1 B and CBV-3 RNA still gave a signal with this primer pair. One disadvantage of primer pair A is, however, that it does not distinguish between rhino- and enteroviruses. This problem could be overcome by using HRV-specific probes for hybridization as described previously (Halonen et al., 1995). On the other hand, primer pair C could be used, particularly when HRV etiology is suspected in some unusual syndromes, although it is less sensitive. Using primer pair A there should be few if any false negatives, since the primers show perfect complementarity with selected rhino- and enterovirus serotypes as Fig. 2 shows. The fact that primer pair D showed lower sensitivity and resulted in a non-specific amplification product with HRV-14 may be due to the mismatches found with the antisense primer (5 - ) in different virus serotypes (Fig. 2). The poor sensitivity may also be partly due to the large size of the amplicon obtained with this primer pair. It may also be assumed that the repeated freezethaw cycles of the specimens in the comparison of primer pairs would lead to the degradation of HRV RNA. This could primarily decrease the sensitivity of primer pair D, which has a relatively long target RNA sequence compared with the other primer pairs studied here. In general, the low sensitivity of some of the primers used could depend on the rhinovirus type in the sample. The reason why selected enterovirus serotypes (CAV-16, -21, CBV-3 and PV-3) were positive, i.e.
they resulted in about 200 bp amplicon with primer pair B, remains to be determined. Since the antisense primer (4 - ) of this pair does not distinguish between rhino- and enteroviruses, the problem must be in the HRV-specific sense primer (RSPE +). In fact, regions in the enterovirus genome close to the primer site exhibit complementarity of five nucleotides with the 3’ end of the primer, and this may be sufficient for amplification when cell culture materials containing high quantities of HRV RNA are used. In conclusion, we describe optimization of a rapid and sensitive RT-PCR test for HRVs, which is superior to the current culture method in sensitivity and rapidity and could eventually replace the latter in the routine diagnosis of HRVs. Our test can be completed during one working day and thus provides a specific diagnosis in the acute phase of the illness, which is important for the prevention of unnecessary use of antibiotics, particularly in lower respiratory tract infections. On the other hand, rapid diagnostic assays may become important for the optimal use of candidate anti-rhinoviral drugs in the future. Owing to the better sensitivity of this test, the possible role of HRVs in various conditions other than the common cold could also be explored further.
Acknowledgements
We thank Anita Arola and Olli Ruuskanen for critical comments, Marita Maaronen for excellent technical assistance and Mikko Arola for clinical samples. This study was supported by grants from the Academy of Finland and the Sigrid Juselius Foundation.
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